Genetic compartmentalization in the complex plastid of...

Genetic compartmentalization in the complex plastid of

Amphidinium carterae

and The endomembrane system (ES) in Phaeodactylum tricornutum

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften

(Dr. rer. nat.)

Vorgelegt dem Fachbereich Biologie der Philipps-Universität Marburg

FB 17 – Biologie, Zellbiologie

von

Xiaojuan Liu

aus Fujian, China Marburg/Lahn 2015

Vom Fachbereich Biologie der Philipps-Universität Marburg

als Dissertation angenommen am: 2015

Erstgutachter: Prof. Dr. Uwe-G. Maier

Zweitgutachterin: Prof. Dr. Ralf Jacob Prof. Dr. Andrea Maisner Prof. Dr. Susanne Önel

Tag der Disputation am: 2015

Publications

X. liu, F. Hempel, S. Stork, K. Bolte, D. Moog, T. Heimerl, UG. Maier, S. Zauner (2015). Addressing various compartments via sub-cellular marker proteins of the diatom model organism Phaeodactylum tricornutum. Algal Research. In preparation.

In preparation:

X. liu, C. Grosche, UG. Maier, S. Zauner (2015). Isolation of individual minicircles via a novel transposon-insertion based approach in Amphidinium carterae. Forthcoming.

Contents

Summary ................................................................................................................................................. 1

Zusammenfassung ................................................................................................................................... 2

Abbreviations .......................................................................................................................................... 3

Figures and Tables ................................................................................................................................... 4

1 Introduction .......................................................................................................................................... 6

1.1 The evolution of complex plastids ............................................................................................. 6

1.1.1 The primary endosymbiosis ........................................................................................... 6

1.1.2 The secondary endosymbiosis........................................................................................ 7

1.1.3 Plastid genome and gene transfer ............................................................................... 10

1.3 Endomembrane system ........................................................................................................... 11

1.3.1 The endomembrane system ......................................................................................... 11

1.3.2 Vacuolar protein transport within endomembrane system ........................................ 12

1.3.2.1 Vacuolar sorting determinants .......................................................................... 13

1.3.2.2 Vacuolar protein transport via vesicles ............................................................. 14

1.3.3 The biosynthesis of N-Glycoproteins on endomembrane system ............................... 17

1.3.4 Tonoplast intrinsic proteins (Tips) ................................................................................ 18

1.3.5 Vacuolar-type H+-ATPases (V-ATPase) ......................................................................... 19

2. Aim..................................................................................................................................................... 21

3 Results ................................................................................................................................................ 22

3.1 Genetic compartmentalization in the complex plastid of Amphidinium carterae .................. 22

3.1.1 The enrichment and isolation of minicircles ................................................................ 22

3.1.2 Analysis of individual minicircles .................................................................................. 23

3.1.2.1 The overview of all individual minicircles .......................................................... 24

3.1.2.2 The core regions of minicircles in A. carterae CCAM0512 ................................ 27

3.1.2.3 Transcription and RNA editing analyses of individual minicircles ..................... 28

3.1.3 Evolution analyses of four A. carterae strains’ minicircles........................................... 29

3.1.3.1 Overall genome characteristics and open reading frames................................ 29

3.1.3.2 Phylogenetic analysis of psbA genes for 15 minicircles of dinoflagellates........ 31

3.1.3.3 Alignment analysis of core regions from four A. carterae strains ..................... 32

3.1.3.4 Phylogenetic analysis of partial LSU/SSU rDNA ................................................. 34

3.2 The endomembrane system (ES) in Phaeodactylum tricornutum .......................................... 37

3.2.1 Identification of marker proteins ................................................................................. 37

3.2.2 Tonoplast intrinsic proteins (Tips) ................................................................................ 37

3.2.2.1 In vivo-localization of Tip1 ................................................................................. 38


3.2.2.3 In vivo-localization of Tip3 and Tip5 .................................................................. 40


3.2.3 In vivo localization of Golgi proteins ............................................................................ 44

3.2.4 In vivo localization of retromer complex ...................................................................... 45

3.2.5 In vivo localization of vacuolar H+-ATPase proteins ..................................................... 47

4 Discussion ........................................................................................................................................... 50

4.1 Genetic compartmentalization of peridinin-containing dinoflagellates ................................. 50

4.1.1 Minicircles of the peridinin-containing dinoflagellate Amphidinium carterae

CCAM0512 ............................................................................................................................. 50

4.1.1.1 Minicircles with coding genes ........................................................................... 51

4.1.1.2 Empty minicircles .............................................................................................. 54

4.1.2 The evolutional relationship of minicircles .................................................................. 55

4.2 “The endomembrane system (ES) in Phaeodactylum tricornutum” ....................................... 57

4.2.1 Identification of tonoplast intrinsic proteins (Tips) ...................................................... 57

4.2.2 Identification of Golgi-marker proteins ........................................................................ 61

4.2.3 Identification of retromer complex proteins................................................................ 62

4.2.4 Identification of vacuolar type H+-ATPases .................................................................. 64

5 Materials and Methods ...................................................................................................................... 65

5.1 Materials .................................................................................................................................. 65

5.1.1 Instruments .................................................................................................................. 65

5.1.2 Membranes and filters ................................................................................................. 66

5.1.3 Antibodies..................................................................................................................... 66

5.1.4 Chemicals ...................................................................................................................... 66

5.1.5 Enzymes ........................................................................................................................ 66

5.1.6 Software and bioinformatic applications ..................................................................... 67

5.1.7 DNA and protein markers ............................................................................................. 67

5.1.8 Oligonucleotide primers ............................................................................................... 67

5.1.9 Vectors .......................................................................................................................... 68

5.1.10 organisms ................................................................................................................... 68

5.2 Methods .................................................................................................................................. 68

5.2.1 Culture of E.coli TOP10 ................................................................................................. 68

5.2.2 Culture of Phaeodactylum tricornutum CCAP 1055/1.................................................. 69

5.2.3 Culture of Amphidinium carterae CCAM0512 .............................................................. 69

5.2.4 Nucleic acid analytics .................................................................................................... 70

5.2.4.1 Plasmid isolation from E.coli ............................................................................. 70

5.2.4.2 DNA and RNA isolation from P. tricornutum ..................................................... 70

5.2.4.3 cDNA synthesis via reverse transcription (RT) .................................................. 71

5.2.4.4 Polymerase chain reaction (PCR) ...................................................................... 71

5.2.4.5 Agarose gel electrophoresis .............................................................................. 72

5.2.4.6 Sequencing ........................................................................................................ 73

5.2.4.7 Restriction and ligation ..................................................................................... 73

5.2.4.8 Transformation of E.coli .................................................................................... 74

5.2.4.10 Transfection of P. tricornutum ........................................................................ 74

5.2.4.11 Minicircles enrichment and isolation from A. carterae CCAM0512 ................ 75

5.2.4.12 Transposon-insertion based approach ............................................................ 75

5.2.4.13 DNA extraction, amplification, and sequencing of LSU rDNA domain D1-D6

and SSU rDNA for A.carterae ......................................................................................... 77

5.2.4.14 Sequence alignment and phylogenetic analyses ............................................. 77

5.2.5 Protein analytics ........................................................................................................... 78

5.2.5.1 Protein isolation from P. tricornutum ............................................................... 78

5.2.5.2 Protein extraction fractionation via carbonate extraction ............................... 78

5.2.5.3 TCA protein precipitation .................................................................................. 79

5.2.5.4 Determination of protein concentration via Amido black ................................ 79

5.2.5.5 SDS-polyacrylamide gel electrophoresis (PAGE) ............................................... 80

5.2.5.6 Western blot analysis ........................................................................................ 80

5.2.5.7 Self-assembling GFP .......................................................................................... 81

5.2.5.8 Construction of eGFP fusion proteins ............................................................... 82

5.2.5.9 Confocal laser scanning microscopy .................................................................. 82

5.2.5.10 Electron microscopy ........................................................................................ 82

6 References .......................................................................................................................................... 84

7 Supplements ..................................................................................................................................... 107

7.1 Open reading frames with no known homology ................................................................... 107

7.2 Identified marker proteins in P. tricornutum ........................................................................ 108

7.3 The Tip2 eGFP fusion protein ................................................................................................ 108

7.4 Sequences of all used oligonucleotides ................................................................................. 109

8 Acknowledgements .......................................................................................................................... 112

9 Curriculum vitae ............................................................................................................................... 113

10 Erklärung......................................................................................................................................... 114

Summary

1

Summary

Peridinin-containing dinoflagellates are important members of single-celled eukaryotic algae, which

arose from an engulfment of an ancient red alga by a so far undefined host cell, a process called

secondary endosymbiosis. Their plastids feature a unique membrane architecture and are

surrounded by only three membranes. As the reduction of the endosymbiont’s genome and gene

transfer from the plastid to the nucleus, the whole plastid genome was reorganized into minicircles

coding for genes normally coded on the plastid genome. In order to isolate individual minicircles

from one representative peridinin-containing dinoflagellate Amphidinium carterae CCAM0512 a

novel transposon-based approach was carried out within this thesis. 89 minicircles were therefore

isolated from A. carterae, 18 (20.2 %) are gene-containing minicircles, 71 (79.8 %) are empty

minicircles. The 18 gene-containing minicircles are divided into three groups of minicircles, six single-

gene minicircles, one two-genes minicircle and one three-genes minicircle. The 71 empty minicircles

are divided into six groups. The characteristics of these minicircles and unique features were

analyzed in this thesis. In contrast to previously reported organellar RNA editing in peridinin-

containing dinoflagellates, no RNA editing was observed on transcripts of minicircles of A. carterae

based on the analysis of coding genes. Additionally, the transcription of open reading frames was

shown in so-called empty minicircles. Finally, based on the comparison with minicircles and rDNA

sequences of three other A. carterae strains, it was speculated that minicircles undergo a rapid

evolutionary diversification.

The mechanisms of protein (e.g. vacuolar proteins) transport and sorting have been well-studied in

plants, yeast and animals. However, little is known in the diatom P. tricornutum. In order to

investigate the protein transport and sorting in P. tricornutum, essential marker proteins have to be

established. In this work, the identification of marker proteins in the endomembrane system was

based on a combination of in silico search for homologous proteins of P. tricornutum to proteins

with known localizations in plants and subsequent in vivo localization studies in P. tricornutum.

Several markers for different subcellular compartments were identified including the plasma

membrane, two vacuolar-like structures, cER, hER, the nuclear envelope and the second outermost

membrane of the complex plastid (PPM). Furthermore, the three parts of the Golgi apparatus and

the cytosol could also be marked. These useful subcellular marker proteins are a very important

prerequisite for studying the mechanisms of protein transport and sorting in P. tricornutum.

Zusammenfassung

2

Zusammenfassung

Peridinin-haltige Dinoflagellaten sind wichtige Vertreter einzelliger eukaryoter Algen, welche durch

die Aufnahme einer anzestralen Rotalge durch eine bislang unbekannte Wirtszelle entstanden sind,

ein als sekundäre Endosymbiose bezeichneter Prozess. Sie besitzen Plastiden mit einer einzigartigen

Membranarchitektur und sind nur von drei Membranen umgeben. Durch die Reduktion des

Endosymbionten-Genoms und den Transfer von plastidären Genen in den Zellkern des Wirtes ist das

plastidäre Genom in Form von sogenannten „minicirclen“ organisiert, auf welchen Gene kodiert sind,

die normalerweise im Plastidengenom kodiert sind. Im Rahmen dieser Arbeit wurden individuelle

„minicircle“ aus dem repräsentativen peridinin-haltigen Dinoflagellaten Amphidinium carterae

CCAM0512 mittels einer neuartigen Transposon-basierten Methode isoliert. Insgesamt konnten

dadurch 89 „minicircle“ isoliert werden, davon kodierten 18 (20.2 %) genetische Informationen

wohingegen 71 (79.8 %) sogenannte „leere minicircle“ waren. Diese 18 kodierenden

„minicircle“ ließen sich in drei Gruppen unterteilen. Sechs „minicircle“ kodierten ein einzelnes Gen,

ein „minicircle“ welcher zwei Gene kodiert sowie ein „minicircle“ welcher drei Gene kodiert. Die 71

„leeren minicircle“ ließen sich in sechs Gruppen einteilen, deren Charakteristika und Eigenschaften

im Rahmen dieser Arbeit analysiert wurden. Analysen der kodierenden „minicircle“ zeigten, dass auf

Transkriptebene keine RNA Edierung in A. carterae beobachtet werden konnte, im Gegensatz zu

„minicirclen“ anderer peridinin-haltiger Dinoflagellaten, bei denen RNA Edierung nachgewiesen

wurde. Im Falle von „leeren minicirclen“ konnte die Transkription von offenen Leserahmen gezeigt

werden. Basierend auf einem Vergleich von „minicirclen“ und rDNA Sequenzen von drei weiteren A.

carterae Stämmen, wurde spekuliert, dass „minicircle“ einer rapiden evolutionären Diversifikation

ausgesetzt sind.

Mechanismen zum Transport von Proteinen und deren Sortierung (z.B. von vakuolären Proteinen)

sind in Pflanzen-, Hefe- und tierischen Zellen gut untersucht, jedoch in der Diatomee P. tricornutum

größtenteils unbekannt. Um den Transport und die Sortierung von Proteinen in P. tricornutum

untersuchen zu können, müssen initial essentielle Markerproteine etabliert werden. Anhand von in

silico Analysen konnten solche Markerproteine, welche homolog zu pflanzlichen Proteinen mit

bekannter Lokalisation sind, in P. tricornutum identifiziert und auf ihre subzelluläre Lokalisation hin

untersucht werden. Mehrere Markerproteine für verschiedene subzelluläre Kompartimente,

einschließlich der Plasmamembran, zwei vakuolen-ähnlicher Strukturen, des cERs, des hERs, der

Kernhülle, der zweitäußersten Membran der komplexen Plastide, der verschiedenen Teile des Golgi-

Apparats und des Cytosols, wurden identifiziert. Diese nützlichen Markerproteine stellen eine

wichtige Voraussetzung für Studien am Mechanismus von Protein Transport und Sortierung in

P. tricornutum dar.

Abbreviations

3

Abbreviations

aa Amino acid Da Dalton

BLAST Basic Local Alignment Search Tool GnTI N-acetylglucosaminyltransferase I

bps basepairs PCR Polymerase chain reaction

BTS bipartite Targeting Sequence FucT α1,3-fucosyltransferase

CCV Clathrin-coated vesicle DV Dense vesicle

cDNA complementary DNA ATPase1-3 vacuolar type H+-ATPase 1-3

cER chloroplast ER mRFP monomeric red fluorescent protein

COPI/II Coat protein I/II psVSD Protein structure-dependent VSD

EE early endosome VSD Vacuolar sorting determinant

EGT Endosymbiotic gene transfer HGT Horizontal gene transfer

EM endomembrane PM Plasma membrane

ER endoplasmic reticulum LV Lytic vacuole

hER host ER PPM The second outermost membrane of the

complex plastid

EST expressed sequence tag Vps26/29 vacuolar protein sorting 26/29

LE late endosome ssVSD Sequence-specific VSD

MC minicircle MVB Multi vesicular body

PPM periplastidal membrane Tip1-5 tonoplast intrinsic protein 1-5

Pt P. tricornutum eGFP enhanced green fluorescent protein

PVC prevacuolar compartment ctVSD C-terminal VSD

rpm rotations per minute CLSM Confocal laser scanning microscope

RT room temperature SA-GFP Self-assembly GFP

SP signal peptide XylT β1,2-xylosyltransferase

TGN trans Golgi network PSV Protein storage vacuole

Figures and Tables

4

Figures

Fig. 1-1: Model of the evolution of complex plastids of green and red algal origin. ...................... 9

Fig. 1-2: A working model for protein sorting to vacuoles. ........................................................... 15

Fig. 1-3: N-glycosylation pathway during the synthesis of glycoproteins in plants. ..................... 18

Fig. 1-4: Subcellular localization of vacuolar type H+-ATPase (VHA) in plant. ............................... 20

Fig. 3-1: The isolated minicircles of A. carterae CCAM0512. ........................................................ 23

Fig. 3-2: GC content for genes-containing minicircles of A. carterae CCAM0512. ........................ 27

Fig. 3-3: Alignment of the core regions of fourteen minicircles of A. carterae CCAM0512. ......... 27

Fig. 3-4. Transcription of psbA gene and ORFs of three empty minicircles. ................................. 28

Fig. 3-5: GC content of psbA and 23S rRNA-containing minicircles in four A. carterae strains. ... 30

Fig. 3-6. Phylogenetic tree analysis based on amino acid sequences encoded by psbA gene from

15 minicircles of dinoflagellates. ........................................................................................... 31

Fig. 3-7. Alignment of the core region of different A. carterae strains’ minicircles. ..................... 32

Fig. 3-8. Alignment of the non-coding regions of four different A. carterae strains’ minicircles. 33

Fig. 3-9. Phylogenetic tree analysis based on LSU rDNA sequences covering domains D1-D6. ... 35

Fig. 3-10. Phylogenetic tree analysis based on SSU rDNA sequences. .......................................... 36

Fig. 3-11: In vivo localization of Tip1-eGFP. ................................................................................... 39


Fig. 3-13: In vivo localization of Tip3- and Tip5-eGFP. ................................................................... 41

Fig. 3-14: In vivo co-staining and co-expression analyses of Tip3 and Tip5 proteins. ................... 42


Fig. 3-16: In vivo localization of proteins located in the Golgi apparatus. .................................... 45

Fig. 3-17: In vivo localization of retromer complex proteins......................................................... 47

Fig. 3-18: Co-expression of retromer complex proteins with Golgi markers. ............................... 47

Fig. 3-19: In vivo localization of V-ATPase proteins. ...................................................................... 49

Fig. 4-1: Schematic overview of identified marker proteins in diatom P. tricornutum. ................ 60

Fig. 5-1: Schematic depiction of transposon-insertion based isolation of minicircles. ................. 77

Fig. S1: The alignment of Tip2 nucleotide sequences in P. tricornutum. .................................... 109

Figures and Tables

5

Tables

Table 3-1: Statistic analysis of isolated individual minicircles. ...................................................... 23

Table 3-2: Properties of minicircles containing chloroplast genes in A.carterae CCAM0512. ...... 24

Table 3-3: Properties of empty minicircles in A.carterae CCAM0512. .......................................... 25

Table 3-4: Identical open reading frames in A. carterae CCAM0512. ........................................... 26

Table 3-5: Reported minicircular sequences in four A.carterae strains. ....................................... 29

Table 3-6: The open reading frames of empty minicircles in A. carterae strains. ........................ 31

Table 3-7: Predicted subcellular localized Tonoplast intrinsic proteins in P. tricornutum. ........... 38

Table 3-8: Predicted subcellular localized Golgi marker proteins in P. tricornutum. .................... 44

Table 3-9: Predicted subcellular localization of retromer complex proteins in P. tricornutum. ... 46

Table 3-10: Predicted subcellular localization of V-ATPase proteins in P. tricornutum. ............... 48

Table S1: All open reading frames mentioned in this study........................................................ 107

Table S2: Proteins localized as eGFP/ mRFP fusions in this study. .............................................. 108

Introduction

6

1 Introduction

1.1 The evolution of complex plastids

1.1.1 The primary endosymbiosis

One of the important steps in the origin of life was the evolution of oxygenous photosynthesis by

ancestral cyanobacteria. The oxygenous photosynthesis in eukaryotes occurs on a photosynthetic

organelle called plastid which arose via a process known as primary endosymbiosis (Marin, Nowack

et al. 2005).

Primary endosymbiosis describes the process in which an ancestral free-living photosynthetic

cyanobacterium was engulfed by a heterotrophic eukaryotic cell. During the co-evolution of the host

cell and the endosymbiont the endosymbiont became an organelle called primary plastid (Deusch,

Landan et al. 2008, Ochoa de Alda, Esteban et al. 2014). Primary plastids are surrounded by two

membranes. The outer membrane acquired prokaryotic and eukaryotic characteristics during its

evolution (Maier, Douglas et al. 2000, Stoebe and Maier 2002). This primary endosymbiosis gave rise

to the three major groups glaucophytes, rhodophytes (red algae), and the chlorophytes (green algae

and land plants) (Fig.1-1) (Cavalier-Smith 1998, Stoebe and Maier 2002, Adl, Simpson et al. 2005,

Reyes-Prieto, Moustafa et al. 2008). Phylogenetic and gene cluster analysis suggests that these

primary plastids are of monophyletic origin (Cavalier-Smith 2000, Martin, Rujan et al. 2002, Reyes-

Prieto, Hackett et al. 2006, Rockwell, Lagarias et al. 2014). Recently, there is new evidence

suggesting that photosynthetic chromotophores in the cercozoan amoeba Paulinella chromatophora

derived from a different cyanobacterium (Bodył, Mackiewicz et al. 2010). This suggests that those

chromotophores originated from an independent endosymbiosis (Bodył, Mackiewicz et al. 2010,

Bodyl, Mackiewicz et al. 2012, Nowack and Grossman 2012).

During their evolution plastid genomes were greatly reduced. Based on comparison with the

genome of free-living cyanobacteria (≥1.6 Mb in size) primary plastids have only about 100 - 200 kbp

(Martin and Herrmann 1998, Stegemann, Hartmann et al. 2003, Reyes-Prieto, Moustafa et al. 2008).

Through a natural and omnipresent process called endosymbiotic gene transfer (EGT) many genes of

the symbiont’s genome were transferred into the host nucleus (Martin and Herrmann 1998,

Stegemann, Hartmann et al. 2003, Reyes-Prieto, Hackett et al. 2006, Reyes-Prieto, Moustafa et al.

2008). Martin et al. found that about 18% of the protein-coding genes in the nucleus originated from

a cyanobacterium (Martin, Rujan et al. 2002). This is a hint that genes were transferred from the

plastid to the host nucleus. Due to the EGT important host nuclear–encoded proteins have to be

retargeted back to the plastids to assist important machineries and metabolic functions. For

Introduction

7

example more than four hundred nuclear-encoded proteins were transferred back to the plastid in

the glaucophytes Cyanophora paradoxa (Facchinelli, Pribil et al. 2013).

1.1.2 The secondary endosymbiosis

Primary plastids subsequently spread by secondary endosymbiosis (Deusch, Landan et al. 2008,

Gould, Waller et al. 2008, Lane and Archibald 2008). Secondary or complex plastids arose by the

uptake of a photosynthetic eukaryotic cell that evolved by primary endosymbiosis into a second

eukaryotic cell (Cavalier-Smith 1998, Cavalier-Smith 2002).

Both green and red algae have been involved in secondary endosymbiotic events (Keeling 2009,

Keeling 2013). The plastid evolution of the secondary emdosymbiosis is strongly debated. The

Cabozoa hypothesis explains that secondary plastids of green algal origin trace back to a single

common endosymbiosis (Cavalier-Smith 1999). However, this hypothesis was contradicted by other

studies analyzing the phylogeny of plastid-encoded proteins. They suggested that these green

plastids were acquired twice independently (Cavalier-Smith 2002, Yang, Elamawi et al. 2005, Green

2011). All red algal derived taxa contain chlorophyII c and are usually referred to as

“chromalveolates”. Haptophytes, heterokonts, apicomplexans, cryptophytes and dinoflagellates are

the major lineages in the chromalveolate group (Fig. 1-1). It has been proposed that one single

secondary endosymbiosis with a red alga gave rise to the common ancestor of all chromaveolates

(Cavalier-Smith 1999, Green 2011). This proposal is known as the chromalveolate hypothesis. Two

chromalveolate genes (fructose bisphosphate aldolase (FBA) and glycerol-3-phosphate

dehydrogenase (GAPDH)) have their unique evolutionary history, which supported the monophyletic

origin of chromalveolates (Fast, Kissinger et al. 2001, Harper and Keeling 2003, Patron, Rogers et al.

2004). The single origin proposal is also supported by the plastid pigmentation in photosynthetic

members, plastid gene and genome relationships, large multigene phylogenies of a concatenated

16-protein data set, an analysis using a 143-protein data set and the unity of SELMA-dependent

protein import (Rodriguez-Ezpeleta, Brinkmann et al. 2005, Hackett, Yoon et al. 2007, Teich, Zauner

et al. 2007, Burki, Inagaki et al. 2009, Hampl, Hug et al. 2009, Zimorski, Ku et al. 2014, Gould, Maier

et al. 2015). But the chromalveolate hypothesis remains very controversial. Some studies based on

genome-analysis are not consistent with this suggested monophyletic origin of the chromalveolates

(Lane and Archibald 2008, Dagan and Martin 2009, Moustafa, Beszteri et al. 2009, Dorrell and Smith

2011). There are different datas indicate that the host cells are not monophyletic. Such as it was

shown that heterokonts are more closely related to alveolates than to haptophytes and

cryptophytes on trees, gene replacement in the plastid genomes of the defined monophyletic

groups, one-third of the proteins in the biosynthetic pathway of carotenoids in chromists are closely

Introduction

8

related to green algal homologs via phylogenetic analyses (Frommolt, Werner et al. 2008). The origin

of red complex plastids is still debating. Recently, Petersen et al. suggested the rhodophycean origin

for the complex plastid Chromera velia, which departs from a single origin of the red complex plastid

(Petersen, Ludewig et al. 2014).

In comparison to primary plastids, secondary complex plastids are surrounded by additional

membranes (Maier, Douglas et al. 2000, Gould, Waller et al. 2008). In the case of organisms with a

green algal endosymbiont, the plastids can be surrounded by three (euglenophytes) or four

membranes (chlorarachniophytes). Important members with a red algal endosymbiont are

haptophytes, heterokonts, cryptophytes and apicomplexa, all of them with secondary plastids

surrounded by four membranes (Stork, Lau et al. 2013). Melkonian suggested that the outermost

membrane is an autophagosomal membrane (Melkonian 1996). The outermost membrane of

complex plastids is also thought to be homologous to a phagocytotic membrane of the host

(Cavalier-Smith 2000, Bolte, Bullmann et al. 2009). After fusion with the endoplasmic reticulum (ER),

the phagocytotic membrane became the chloroplast ER membrane (cER) in heterokonts,

haptophytes and cryptophytes (Lemgruber, Kudryashev et al. 2013). The second outermost

membrane of complex plastids, also called periplastidal membrane (PPM), is thought to be

homologous to the cytoplasmic membrane of the endosymbiont (Cavalier-Smith and Chao 2003). It

was also suggested that the host ER membrane was involved to form the two outermost membranes

(Zimorski, Ku et al. 2014, Gould, Maier et al. 2015). The two innermost membranes of complex

plastids appear to originate from the inner and outer envelope of primary plastids (Schleiff and

Becker 2011, Stork, Lau et al. 2013). However, a different situation is found in peridinin-containing

dinoflagellates the complex plastids of red algal origin are surrounded by only three membranes

(Cavalier-Smith 2000). The evolution of dinoflagellates is described in detail in the next paragraph. In

chromalveolate lineages cryptophytes are the only member demonstrated to have retained the

nucleomorph (the former nucleus of the engulfed red alga) between the outer and inner chloroplast

membrane pair (Archibald 2007).

Introduction

9

Fig. 1-1: Model of the evolution of complex plastids of green and red algal origin.

During primary endosymbiosis an ancestral free-living photosynthetic cyanobacterium was engulfed by a eukaryotic cell

and established as an endosymbiont in the three lineages glaucophyta, rhodophyta and chlorophyta. Engulfment of a

green or red alga by another unicellular eukaryote and subsequent reduction of the symbiont to an organelle led to the

development of so-called secondary or complex plastids. Two independent secondary endosymbiotic events involving

algae of the chlorophyta and different eukaryotic hosts resulted in the chlorarachniophytes (1) and euglenophytes (2). The

red algal derived taxa collectively are usually referred to as “chromalveolates”. Haptophytes, heterokonts, apicomplexans,

cryptophytes and dinoflagellates are the major lineages in the chromalveolate group, but this is strongly discussed. It is

unknown if red secondary plastids are mono- or polyphyletic. (Modified after Zimorski (Zimorski, Ku et al. 2014)).

Dinoflagellates are an extremely diverse group of photosynthetic species and non-photosynthetic

species. Peridinin-containing dinoflagellates are distinguished from the other species in

dinoflagellates by the pigment peridinin and three-membrane surrounded plastids (Dodge and Lee

2000). During the evolution one of the ancestral four membranes must have been lost in

dinoflagellates (Zhang, Green et al. 1999, Barbrook and Howe 2000, Zhang, Green et al. 2000, Stoebe

and Maier 2002). Gould et al. suggested the plastids have probably lost the second outermost of

four membranes (Gould, Maier et al. 2015). It was shown that the membranes of the peridinin-

containing plastid are not connected to the host ER and the outermost membrane does not have

ribosomes (Gould, Maier et al. 2015).

Introduction

10

The evolutionary origin of peridinin-containing plastids in dinoflagellates is also highly debated

because of the reduced number of plastid membranes. Previous studies suggested that the

peridinin-containing plastids arose from secondary endosymbiosis (Mcfadden 2001, Grosche,

Hempel et al. 2014, Gruber, Rocap et al. 2015). However, several hypothesizes indicate that the

plastids of dinoflagellates evolved via a more complicated tertiary endosymbiosis, where a

secondary plastid-containing alga was engulfed and reduced to an organelle (Hackett, Yoon et al.

2004, Keeling 2010, Gabrielsen, Minge et al. 2011, Burki, Imanian et al. 2014). Dinoflagellates have

been shown to contain heterkontophyte-, cryptophyte- and haptophyte-derived tertiary plastids in

the so-called “Dinotoms”, Dinophysis and Kareniaceae groups, respectively (Burki, Imanian et al.

2014).

1.1.3 Plastid genome and gene transfer

More than 25 years ago the first complete sequence of a plastid genome was reported in the

liverwort Marchantia polymorpha (Ohyama, Fukuzawa et al. 1986). There are more and more plastid

genomes available now, including those of land plants and algae. The plastid genomes of algae and

land plants usually are single circular DNA molecules with a size of 100 - 200 kbp and contain

approximately 100 - 250 genes (Barbrook and Howe 2000, Nisbet, Hiller et al. 2008). Encoded on

these genomes are many components of the photosynthesis, chloroplast replication and protein

synthesis machinery (Hiller 2001).

In contrast to the common genome organization found in most eukaryotic groups harboring plastids,

peridinin-containing dinoflagellates have a unique degenerated and rearranged plastid genome

(Zhang, Green et al. 1999, Barbrook and Howe 2000, Laatsch, Zauner et al. 2004, Takishita, Ishida et

al. 2004). After the disintegration and reconfiguration of their plastid genome, the genes are located

to plasmid-like molecules of a size of about 0.4 - 10kbp instead of a conventional plastid

chromosome genome. These molecules are called minicircles (Zhang, Green et al. 1999, Barbrook

and Howe 2000, Howe, Nisbet et al. 2008). Each minicircle contains one to three genes as well as a

non-coding region. Most encoded proteins are related to photosynthesis. Additionally many ‘empty’

minicircles have been identified. Although they contain a number of open reading frames of

different lengths no significant homology could be detected, thus leaving their purpose unknown

(Hiller 2001, Nisbet, Koumandou et al. 2004). Based on the alignment of the non-coding regions of

all known minicircles within species a highly conserved region called core region was found. The core

region is thought to be responsible for the initiation of replication, the maintenance of the copy

number and/or the transcription of minicircles (Zhang, Green et al. 1999, Barbrook and Howe 2000,

Hiller 2001, Zhang, Cavalier-Smith et al. 2002, Barbrook, Dorrell et al. 2012). Additionally to the core

Introduction

11

and coding region minicircles contain a non-coding region. It has been shown that the non-coding

regions possess a tripartite conserved sequence that could be folded in silico into secondary

structures (Howe, Nisbet et al. 2008). Only about 15 genes have been found on dinoflagellate

minicircles (varying on species) until now (Zhang, Green et al. 1999, Barbrook and Howe 2000, Hiller

2001, Nisbet, Koumandou et al. 2004, Howe, Nisbet et al. 2008). Compared to the size of other

plastid genomes the plastid genome of dinoflagellates is undoubtedly the smallest (Green 2004,

Green 2011). This shrunken plastid genome might result from the missing of numerous genes. Some

authors suggested that these missing genes were either deleted or transferred to the host genomes

during the process of plastid acquisition (Bachvaroff, Concepcion et al. 2004, Green 2004, Hackett,

Yoon et al. 2004, Bachvaroff, Sanchez-Puerta et al. 2006).

As already mentioned during the previous endosymbiosis as well as the following (secondary or even

tertiary) endosymbiosis there was a massive transfer of endosymbiotic genes to the host nucleus

known as EGT.

However, the reduced plastid genome is insufficient to meet the wide variety of plastid functions

such as photosynthesis, lipid and protein biosynthesis. Because of this, the proteins had to be

somehow targeted to the plastids. It was shown that nucleus-encoded proteins required for plastidal

functions were transported back to the plastids with the aid of targeting signals and a protein-import

machinery (Martin and Herrmann 1998, Weber, Linka et al. 2006).

1.3 Endomembrane system

1.3.1 The endomembrane system

In eukaryotic cells the endomembrane system is made up of several functionally different organellar

membranes, including the nuclear envelope, ER, Golgi apparatus, lysosomes or vacuoles, different

vesicles (more detail in 1.3.2.2) and the plasma membrane (PM) (Fig.1-2). These organelles are

located in the cytoplasm and interconnected by vesicular transport (Schellmann and Pimpl 2009).

This membrane system divide the cell into functional and structural compartments (Galili 2001,

Gautreau, Oguievetskaia et al. 2014) . It is necessary for proteins to be transported to the right

destinations.

The endomembrane compartments fulfill specific functions in the transport of proteins. The

endoplasmic reticulum (ER) has a central role in producing, processing and transporting proteins.

The Golgi apparatus is another important organelle. The Golgi consists of three networks, cis-Golgi

network mainly receive proteins from ER, medial-Golgi transport proteins from cis-Golgi to trans

Golgi, while the trans-Golgi or trans Golgi network (TGN) send proteins to next organelles. During

Introduction

12

the transport of proteins from the ER to the Golgi apparatus, proteins are modified, sorted and

finally transported to destinations such as the vacuole, the extracellular space (so called anterograde

transport) or recycled back to the Golgi apparatus (so called retrograde transport) (Bolte, Brown et

al. 2004). Endosomes are single membrane-surrounded compartments and represent a major and

important sorting compartment in the endomembrane system. Endosomes can be classified into

early endosomes, late endosomes and recycling endosomes depending on their main functions

(Reyes, Buono et al. 2011). But in most cases it is difficult to distinguish these endosomes (Otegui

and Spitzer 2008). The trans-Golgi network (TGN) is believed to serve as an early endosome (Mallard,

Tang et al. 2002, Dettmer, Hong-Hermesdorf et al. 2006, Viotti, Bubeck et al. 2010). The main

function of TGN/early endosomes is to mediate vacuolar protein transport, receive internalized

proteins from the plasma membrane or retransport endocytosed proteins back to the plasma

membrane and recycle protein sorting receptors (here also called recycling endosomes) (Mallard,

Tang et al. 2002, Otegui and Spitzer 2008). Subsequently, early and recycling endosomes are

believed to mature into late endosomes or multi vesicular bodies (MVBs), also called prevacuolar

compartments (PVCs) (Reyes, Buono et al. 2011). Late endosomes have two important functions,

recycling of protein sorting receptors back to the TGN and transporting of newly synthesized

proteins from the Golgi apparatus to vacuoles (Piper and Katzmann 2007, Otegui and Spitzer 2008).

It is obvious that the endosomal network is a key compartment that mediates the concentration of

proteins in the Golgi apparatus, vacuoles and plasma membrane (Tse, Mo et al. 2004, Otegui and

Spitzer 2008).

The most visible compartment surrounded by only one membrane is the vacuole (Maruyama, Mun

et al. 2006, Zouhar and Rojo 2009). Vacuoles are involved in maintaining of internal hydrostatic

pressure and storage of water, ions, nutrients and secondary metabolites. Similar to animal

lysosomes, they also act in intracellular digestion of various waste products and toxic substances

(Matsuoka 1993, Otegui and Spitzer 2008, Pereira, Pereira et al. 2013, Ebine, Inoue et al. 2014). In

plant cells, at least two different types of vacuoles can be found, the central lytic vacuole (LV) for

protein degradation and the protein storage vacuole (PSV) (Swanson, Bethke et al. 1998, Frigerio,

Jolliffe et al. 2001, Frigerio, Hinz et al. 2008).

1.3.2 Vacuolar protein transport within endomembrane system

Proteins that are secreted to the extracellular space or retained in the endosomal membrane system

are commonly called secretory proteins (Vitale and Hinz 2005). Most secretory proteins contain an

ER signal peptide or a hydrophobic transmembrane domain for insertion into the ER membrane

(Johnson and van Waes 1999, Xiang, Etxeberria et al. 2013). When the signal peptide is cleaved off

Introduction

13

from the proteins and then other specific targeting signals are used for transporting proteins to the

destinations (Jürgens 2004, Xiang, Etxeberria et al. 2013, Xiang and Van den Ende 2013). Therefore,

the targeting signals and correct transport routes are important for the targeting of the vacuolar

protein to the final destination.

1.3.2.1 Vacuolar sorting determinants

Protein sorting to the precise destinations depends on many targeting information from the protein.

The identification of vacuolar sorting signals/vacuolar sorting determinants (VSS/VSDs) is necessary

for better understanding of protein targeting to the vacuoles at the post-Golgi level. Three distinct

sorting determinants are known in plant cells. Sequence-specific VSDs (ssVSDs), C-terminal VSDs

(ctVSDs) and protein structure-dependent VSDs (psVSDs) have been reported (Hwang 2008,

Hegedus, Coutu et al. 2015). Without these important signals vacuolar proteins will be targeted to

the wrong subcellular compartments or degraded.

Many studies have shown that sequence-specific VSDs are usually located at the N-terminus of the

proteins and mainly sort them to the lytic vacuoles, such as barley aleurain (Koide, Matsuoka et al.

1999, Sanmartin, Ordonez et al. 2007). In some cases sequence-specific VSDs are also recognized by

vacuolar sorting receptors and guide proteins to the PSVs, such as seed storage protein 2S albumin

and toxin ricin (Frigerio, Jolliffe et al. 2001, Jolliffe, Brown et al. 2004).

C-terminal VSDs are always located at the C-terminal part of the protein. They generally lead

proteins target to the protein storage vacuoles (Nishizawa, Maruyama et al. 2003, Vitale and Hinz

2005, Maruyama, Mun et al. 2006). Finally psVSDs are based on the tertiary (physical) structure of

proteins and usually transport proteins to the protein storage vacuole (Neuhaus and Rogers 1998,

Jolliffe, Brown et al. 2004, Jolliffe, Craddock et al. 2005, Neuhaus 2007, Zouhar and Rojo 2009).

Recently some studies have shown that some proteins contain two different VSDs at the same time.

For example, the existence of a sequence-specific VSD and a ctVSD at the C-terminal region of α'

subunit of soybean β-conglycinin directs the protein to the PSVs (Nishizawa, Maruyama et al. 2003).

The protein cardosin A with a characteristic ctVSD and an untraditional vacuolar sorting domain (PSI)

was transported to the vacuole. Plant specific domain (PSI) is an additional protein domain of about

100 amino acids present in the protein precusors and is normally deleted after maturation (Simoes

and Faro 2004). It was also demonstrated that any one of these two signals alone is sufficient to sort

proteins to the vacuole (Pereira, Pereira et al. 2013).

However, Pompa et al shown that the vacuolar phaseolin is secreted to the apoplast without the C-

terminal tetrapeptide AFVY. When a cysteine residue was added to the phaseolin for forming

interchain disulfide bond, the phaseolin polypeptides connected by engineered disulfide bonds are

Introduction

14

transported to vacuoles again (Pompa, De Marchis et al. 2010). Nishizawa et al. proposed that the

proportion between the size of the protein and the copy number of vacuolar sorting determinants is

also very important for the sorting efficiency (Nishizawa, Maruyama et al. 2003). Based on these

data in protein sorting to the vacuole, it is clear that the three well-known types of VSDs identified

so far is not thoroughly sufficient to explain all the vacuolar targeting mechanisms.

1.3.2.2 Vacuolar protein transport via vesicles

Vacuolar protein transport is mediated by different intermediate vesicles. Previous studies have

shown that most of newly synthesized proteins are transported from ER to the Golgi apparatus via a

coat protein II (COPII)-vesicle-dependent manner (Ritzenthaler, Nebenführ et al. 2002, Takeuchi,

Ueda et al. 2002, daSilva, Snapp et al. 2004, Yang, Elamawi et al. 2005). Different types of ER export

motifs have already been identified for exiting the ER (e.g. a di-basic motif RKR in tobacco) (Barlowe

2003, Hanton, Renna et al. 2005, Lee and Miller 2007, Lee and Miller 2007, Schoberer, Vavra et al.

2009). ER export motifs are recognized by the known cargo-binding site on Sec24 component of

COPII complex, and then the protein will be captured into COPII vesicles and transported to the next

organelle (Miller, Beilharz et al. 2003, Mossessova, Bickford et al. 2003, Lee and Miller 2007). COPI

was identified in mediating retrograde traffic from the cis-Golgi to the ER. Proteins with ER retention

signals are mistargeted to the Golgi. These proteins can be retransported back to the ER in COPI

vesicles (Pimpl, Taylor et al. 2006). It was also shown that COPI adjusts the vesicular traffic pathway

within the Golgi apparatus (Movafeghi, Happel et al. 1999, Contreras, Ortiz-Zapater et al. 2000,

Pimpl, Movafeghi et al. 2000, Pimpl, Hanton et al. 2003).

In plants different routes were used for the vacuolar protein transport after the ER (Fig. 1-2). One of

the possibilities involves the passage through the Golgi apparatus. Previous studies have shown that

transport of some important lytic vacuolar proteins from the Golgi apparatus to the lytic vacuoles

(LVs) is mediated by clathrin-coated vesicles (CCVs) with a diameter of 50 to 70 nm (Hohl, Robinson

et al. 1996, Robinson, Hinz et al. 1998, De Marchis, Bellucci et al. 2013). Specific vacuolar targeting

signals are recognized by protein sorting receptors in the membrane of the TGN (Rouillé, Rohn et al.

2000). Subsequently the protein sorting receptors are recognized by adaptor proteins (Xiang,

Etxeberria et al. 2013, Xiang and Van den Ende 2013). The ligand-receptor complexes are recruited

into the clathrin-coated vesicles (Ahmed, Rojo et al. 2000, Kalthoff, Groos et al. 2002, Happel,

Höning et al. 2004, Song, Lee et al. 2006). CCVs containing proteins bud from the trans Golgi network,

then deliver their cargoes to LVs after fusion with multi vesicular bodies (Fig. 1-2 marked by black

arrow) (Robinson and Bonifacino 2001, Tse, Mo et al. 2004, Van Damme, Gadeyne et al. 2011).

Clathrin-coated vesicles are one major style of vesicles in plant. Clathrin-coated vesicles can also bud

Introduction

15

from the plasma membrane for the uptake of nutrients, the delivery and regulation of signaling

components as well as the recycling of proteins (Maldonado-Mendoza and Nessler 1996, Holstein

2002, McMahon and Boucrot 2011, Reynolds, August et al. 2014).

Transport of lytic vacuolar proteins to the LV can also take an alternative pathway from the ER

bypassing the Golgi, as shown in Fig. 1-2 (marked by purple arrow). This process is mediated by

intermediate compartments, called ER bodies (Matsushima R 2003, Hara-Nishimura, Matsushima et

al. 2004). When the vacuolar sorting motifs are recognized by protein sorting receptors, ligand-

receptor complexes are recruited into the ER bodies. The ER bodies (diameter of less than 1000 nm)

are oil, protein or rubber containing ER-derived membrane structures (Herman and Schmidt 2004,

Herman 2008, Xiang, Etxeberria et al. 2013). Therefore, the lytic vacuole sorting pathway could be

defined as ER → Golgi → CCV → multi vesicular body → LV and ER → ER body → LV pathways.

Fig. 1-2: A working model for protein sorting to vacuoles.

Many different sorting routes are known for proteins transport to vacuoles. During the transport of lytic vacuolar proteins,

proteins are directly targeted from the ER to their destination in ER bodies bypassing the Golgi apparatus (marked by

purple arrow). Proteins are transported from the Golgi apparatus to the lytic vacuole (LV) via prevacuolar compartments or

multivesicular bodies (MVBs). This pathway is mediated in clathrin-coated vesicles (CCVs) (indicated by black arrow).

During the transport of storage proteins, proteins are sorted to protein storage vacuole (PSV) by a receptor-mediated way

(brown heart) in clathrin-coated vesicles (red arrow). Some proteins are transported to PSV by a receptor-mediated

pathway (brown star) via dense vesicles (DVs) (blue arrow). The multi vesicular body (MVB) might be an alternative

intermediate compartment during the protein transport from CCVs and DVs to PSV (yellow arrows). Some proteins bypass

the Golgi apparatus using precursor-accumulating (PAC) vesicles (green arrow). These vesicles also receive proteins from

the Golgi apparatus. PM: plasma membrane. (Modified after Vitale and Hinz 2005)

Introduction

16

However, the targeting of proteins from the ER to the protein storage vacuoles is different and more

complicate. It is majorly mediated by dense vesicles (DVs). Different from the lytic protein transport

the interaction of ligands and receptors is calcium-dependent in storage vacuolar protein transport

(Shimada, Fuji et al. 2003). DVs are small vesicles with a diameter of 150 - 200 nm. Vacuolar proteins

are recruited into the mature DVs released from the TGN. Mature DVs are not involved in a protein

coat (Hohl, Robinson et al. 1996). Finally the DVs deliver their cargoes to the PSVs (marked by blue

arrow) (Vitale and Hinz 2005). All available evidences indicate that some seed storage proteins, such

as most 2S ablumins and the toxin ricin, would also be delivered to protein storage vacuoles via

clathrin-coated vesicles (marked by red arrow) (Vitale 2001, Jolliffe, Brown et al. 2004, Vitale and

Hinz 2005). MVBs or PVCs are the alternative intermediate target of CCVs and DVs during the

transport route from the Golgi apparatus to PSVs (marked by yellow arrow) (Robinson, Hinz et al.

1998, Jiang, Phillips et al. 2000, Tse, Mo et al. 2004) (Fig. 1-2).

Studies have also shown that vacuole residing proteins are sorted directly from the ER to the PSVs

using precursor-accumulating (PAC) vesicles and are therefore transported in a Golgi-independent

way (Hara‐Hishimura, Takeuchi et al. 1993, Vitale 2001, Michaeli, Avin-Wittenberg et al. 2014).

There is an alternative route. Some vacuolar proteins could be transported from the Golgi apparatus

to the PAC vesicles and then target to the PSV (marked by green arrow) (Watanabe, Shimada et al.

2004). Therefore, the protein storage vacuole sorting pathway could be mainly defined as ER →

Golgi → DV/CCV → (multi vesicular body) → PSV and ER → PAC → PSV pathways.

Receptor-mediated sorting pathways for secretory proteins in eukaryotic cells depend on

mechanisms to recycle the receptors after the dissociating of receptor-ligand complexes (Niemes,

Langhans et al. 2010). The recycling of receptors are mediated by different complexes such as

retromer. The retromer is a coat complex that locates on the cytosolic face of the TGN/early

endosome (Vergés, Sebastián et al. 2007, Schellmann and Pimpl 2009, Seaman, Harbour et al. 2009).

In yeast, plants and mammals the retromer complex contains two subcomplexes, a large

subcomplex formed by three cargo selective adaptor subunits (Vps26, Vps29 and Vps35) and a small

subcomplex formed by membrane deforming sorting nexin proteins (SNXs) (Seaman 2004,

Bonifacino and Hurley 2008, Cullen and Korswagen 2012). Most newly synthesized proteins are

transported via a protein sorting receptors-dependent parthway. The protein sorting receptors have

been well-characterized in some organisms such as the seed storage protein receptors in A. thaliana,

vacuolar protein sorting 10 (Vps10) in yeast S. cerevisiae and mannose 6-phosphate receptors (MPR)

in mammals (Horazdovsky, Davies et al. 1997, Nothwehr, Bruinsma et al. 1999, Arighi, Hartnell et al.

2004, Carlton, Bujny et al. 2004, Seaman 2004, Niemes, Langhans et al. 2010, McGough and Cullen

2011, Robinson, Pimpl et al. 2012). In order to maintain the forward transport of proteins the

Introduction

17

efficient retrograde transport of the protein sorting receptor is a critical important step. This

recycling of receptors is mediated by some complexes such as the retromer. Moreover, the retromer

is also responsible for many other physiological and developmental processes such as mediating the

transport of internalized toxin and auxin efflux carriers (more diverse functions of retromer see

Bonifacino and Hurley 2008).

1.3.3 The biosynthesis of N-Glycoproteins on endomembrane system

Glycosylation is a very important translational modification for proteins. There are different

glycosylations such as N-glycosylation, O-glycosylation and phosphor-glycosylation. N-glycosylation

is a major co- and post-translational modification of proteins in eukaryotic cells. It has been

discussed that N-glycosylation plays a crucial role in the folding, assembly, structural formation and

stability of several important proteins (Rayon, Lerouge et al. 1998, Baïet, Burel et al. 2011, Mathieu-

Rivet, Kiefer-Meyer et al. 2014). Moreover previous studies have shown that the glycosylation plays

a role in the sorting of proteins to the vacuoles, especially in the Golgi-independent secretory

pathway (Rayon, Lerouge et al. 1998, Paris, Saint-Jean et al. 2010, Pereira, Pereira et al. 2013,

Pereira, Pereira et al. 2014).

It has been discussed that the majority of secretory proteins are N-glycosylated in the endoplasmic

reticulum (ER) and the Golgi apparatus (Fig. 1-3) (Strasser 2014). The process of N-glycosylation can

be mainly divided into three stages: during the initial phase of the process a precursor

oligosaccharide is synthesized in the ER and transferred en bloc to the protein by a

oligosaccharyltransferase, which is part of a translocation complex (Baiet, Burel et al. 2011, Kajiura,

Okamoto et al. 2012, Bosch, Castilho et al. 2013, Mathieu-Rivet, Kiefer-Meyer et al. 2014), the

resulting N-glycoproteins are transported to the Golgi apparatus and further catalysed and modified

by a large number of highly conserved membrane-bound enzymes, such as glycosyltransferases and

glycosylhydrolases, such as N-acetylglucosaminyltransferase I (GnTI), β1, 2-xylosyltransferase (β1, 2-

XylT), α1, 3-fucosyltransferase (α1, 3-FucT), α1, 4-fucosyltransferase (α1, 4-FucT) and so on (Kornfeld

and Kornfeld 1985, Strasser, Mucha et al. 2000, Wilson 2002, Bondili, Castilho et al. 2006, Strasser,

Bondili et al. 2007, Strasser 2014), finally the mature glycoproteins are either secreted or targeted

into the plasma membrane.

Introduction

18

Fig. 1-3: N-glycosylation pathway during the synthesis of glycoproteins in plants.

The synthesis, en bloc transfer and initial modification of precursor oligosaccharide occur in the ER under the catalytic

action of Glu I/II/III enzymes. Subsequently, the processing and modification of the oligosaccharide chain is performed in

the cis- medial- and trans-Glogi apparatus. At last, the mature glycoproteins are either targeted into the plasma membrane

or secreted. Glu I: glucosidase I, Glu II: glucosidase II, Glu III: glucosidase III, Man: mannosidase, GnTI: N-

acrtylglucosaminyltransferase I, GnTII: N-acrtylglucosaminyltransferase II, β1, 2-XylT: β1, 2-xylosyltransferase, α1, 3-FucT:

α1, 3-fucosyltransferase, α1, 4-FucT: α1, 4-fucosyltransferase, β1, 3-GalT: β1, 3-galactosidase. (Modified after Hyun-Soon K.

Jae-Heung J. et al 2014)

1.3.4 Tonoplast intrinsic proteins (Tips)

Some membrane proteins do not have a direct effect on the protein sorting and transport, but they

are important for the cellular metabolism. Aquaporins are channel proteins belong to the the major

intrinsic proteins (MIPs) superfamily. Aquaporin pore can selectively mediate the transport of water,

gases and small neutral solutes such as urea, glycerol as well as silicic acid (Maurel 1997, Loque,

Ludewig et al. 2005, Lienard, Durambur et al. 2008, Uehlein and Kaldenhoff 2008, Wudick, Luu et al.

2014). In most plant species aquaporins can be subdivided into four groups, the plasma membrane

intrinsic proteins (PIPs), nodulin 26-like intrinsic proteins (NIPs) localized in the plasma membrane

and ER, the tonoplast intrinsic proteins (TIPs) and small basic intrinsic proteins (SIPs) localized in the

Introduction

19

ER (Johansson, Karlsson et al. 2000, Baiges, Schaffner et al. 2002, Quigley, Rosenberg et al. 2002,

Pandey, Sharma et al. 2013).

Aquaporins have been well-studied on the structure and function in plants, yeast and animals

(Kaldenhoff, Ribas-Carbo et al. 2008, Maurel and Plassard 2011, Li, Santoni et al. 2014). Aquaporin

proteins contain six conserved transmembrane-spanning α-helices linked by three extra- and two

hydrophobic intracellular loops (Kaldenhoff and Fischer 2006, Fischer and Kaldenhoff 2008,

Chaumont and Tyerman 2014). The N-terminus and C-terminus of aquaporin proteins are located at

the cytosolic side of the membrane (Johansson, Karlsson et al. 2000, Uehlein and Kaldenhoff 2008).

It has been shown that the first three transmembrane domains and the rest three transmembrane

domains form an inversely repeat (Kaldenhoff, Ribas-Carbo et al. 2008, Chevalier and Chaumont

2014). Previous studies have already shown that the loops play a critical role in the forming of

transmembrane channels and its stability (Werner, Uehlein et al. 2001, Kaldenhoff, Bertl et al. 2007,

Kaldenhoff, Ribas-Carbo et al. 2008). Two conserved asparagine-proline-alanine (NPA) motifs were

located in the different loop regions. The NPA motif and adjacent residues were thought to be

critically important for water transport activity (Chaumont, Barrieu et al. 2001, Park, Scheffler et al.

2010).

1.3.5 Vacuolar-type H+-ATPases (V-ATPase)

The ATPase is an important multi-subunit transmembrane complex (Clarke, Köhler et al. 2002,

Dettmer, Hong-Hermesdorf et al. 2006). There are different classes of ATPases (F-ATPases, V-

ATPases, E-ATPases, P-ATPases and A-ATPases), which can be distinguished by their function,

structure and the transport of ions (McKersie and Bruce 2009, Xi and Wu 2011, Islam, Patwary et al.

2014). It has been shown that V-ATPases are mainly accumulated at vacuoles, ER, Golgi apparatus,

plasma membrane and endosomes (Sze, Schumacher et al. 2002, Krebs, Beyhl et al. 2010)(Fig. 1-4).

V-ATPase complexes contain a cytosolic subcomplex V1 (subunits A-H) and hydrophobic subcomplex

V0 (subunits a, c, c’, c’’, d and e) (Dettmer, Hong-Hermesdorf et al. 2006, Seidel, Schnitzer et al. 2008,

Ma, Qian et al. 2012). The major function of this complex is to pump protons from the cytoplasm

into the lumen of organelles or to the outside of the cell. The V-ATPase catalyzes the hydrolysis of

ATP into ADP and a free phosphate ion (Ma, Qian et al. 2012). During the catalytic process a lot of

energy is released which is used for the cell metabolism including endosomal transports (Dettmer,

Liu et al. 2010, Zhou, Bu et al. 2015). In Puccinellia tenuiflora decreasing of the V-ATPase activity

obstructs endosomal trafficking (Zhou, Bu et al. 2015). It was also shown that the inhibition of VHA

interrupt the transport from TGN to the vacuole as inhibitor concanamycin prevent the TGN

formation and release fron the Golgi apparatus (Scheuring, Viotti et al. 2011).

Introduction

20

Fig. 1-4: Subcellular localization of vacuolar type H+-ATPase (VHA) in plant.

It was shown that vacuolar-type H+-ATPases (VHAs) are mainly distributed to the ER, Golgi apparatus, vesicles, vacuoles

and plasma membrane. The major role of VHAs is to pump H+ into the lumen of organelles or out of the cell and provide

energy for cell metabolites. Abbreviations: AHA, A. thaliana plasma membrane H+-pumping ATPase; AVP1, vacuolar H+-

pumping ppase; CAX1, Ca2+/ H+ antiporter. (Modified from Sze, Schumacher et al. 2002)

Aim

21

2. Aim

Peridinin-containing dinoflagellates are unicellular alveolates which evolved via secondary

endosymbiosis event. The reduction of the endosymbiont genome in addition to gene transfer from

the plastid to the nucleus resulted in a unique endosymbiont genome organization in peridinin-

containing dinoflagellates. Genes normally found on plastid genomes have been organized into so-

called minicircles. Previous studies have already reported several minicircle sequences from

different dinoflagellates (Barbrook and Howe 2000, Hiller 2001, Zhang, Cavalier-Smith et al. 2002,

Moore 2003, Laatsch, Zauner et al. 2004, Nisbet, Koumandou et al. 2004, Barbrook, Santucci et al.

2006, Howe, Nisbet et al. 2008). However, it remains limit on the isolation of the single minicircle

molecule. The aim of this work was to isolate individual minicircle molecules via novel transposon-

based approach from the dinoflagellate A. carterae CCAM0512. Based on these individual minicircle

molecules the characteristics and evolutionary relationship of minicircles were analysed. In order to

investigate the localization of minicircles individual minicircle molecules were manipulated for

retransfection of A. carterae CCAM0512.

Proteins are usually synthesized on the rough endoplasmic reticulum (rER), and are then sorted to

different destinations (e.g. vacuole) through the endomembrane system by the secretory pathway.

The protein transport and sorting mechanisms (such as the sorting routes, targeting signals and

vacuolar protein sorting receptors) have already been well-studied in plant cells, yeast and animals

(Geuze 1995, Bonifacino and Traub 2003, Pereira, Pereira et al. 2013, Xiang, Etxeberria et al. 2013,

Pereira, Pereira et al. 2014, Zhang, Hicks et al. 2014). Little is known about the protein transport and

sorting through the endosomal compartments in diatom P. tricornutum. The aim of this work was to

gain insight into the sorting mechanisms of protein with targeting signals. Defining appropriate

marker proteins for subcellular organelles are essential for studying the mechanisms. However, the

known marker proteins in P. tricornutum are less enough. To extend the dataset of marker proteins

homologous proteins to known localization proteins in plants were identified in P. tricornutum. To

investigate the subcellular localization of candidate marker proteins eGFP- or mRFP-fusion proteins

were studied in vivo localization by confocal laser-scanning microscopy.

Results

22

3 Results

3.1 Genetic compartmentalization in the complex plastid of Amphidinium carterae

Peridinin-containing dinoflagellates are unicellular alveolates that evolved via secondary

endosymbiosis (Mcfadden 2001, Shalchian-Tabrizi, Skånseng et al. 2006). The reduction of the

endosymbiontic genome in addition to gene transfer from the plastid to the nucleus led to a unique

endosymbiont genome organization in peridinin-containing dinoflagellates (Zhang, Green et al. 1999,

Barbrook and Howe 2000, Howe, Barbrook et al. 2003). In these organisms genes normally found on

the conventional plastid genome have been organized into so-called minicircles (Barbrook and Howe

2000, Hiller 2001, Zhang, Cavalier-Smith et al. 2002, Howe, Nisbet et al. 2008). In order to retrieve

their full-length DNA sequence a novel transposon-based approach was used to isolate individual

minicircle molecules from the peridinin-containing dinoflagellate A. carterae CCAM0512.

3.1.1 The enrichment and isolation of minicircles

It was already shown by Laatsch et al. that a high quantity of minicircles could be enriched and

isolated by the alkaline lysis (Laatsch, Zauner et al. 2004). Here in order to enrich and isolate

minicircles from A. carterae CCAM0512, additionally to the alkaline lysis different suppliers for

alkaline lysis based kits were tried (see material and method 5.2.4.11). It was found that a significant

quantity of minicircles could only be enriched and isolated via the alkaline lysis but not alkaline lysis

based kits. Koumandou and Howe have already shown that the copy number of different minicircles

per cell is low during the exponential growth stage, but the number is increasing during the later

growth phase (Koumandou and Howe 2007). It was found that the significant minicircles could only

be visible on an agarose gel via the enrichment and isolation from the old cultures about four to five

weeks but not younger cultures.

The isolation of minicircles was shown on Fig. 3-1. A strong signal was detectable at a size of about

2000 bps (lane 1 and lane 2), this resulted from the isolated minicircles. The gel of red box area was

retrieved and used for the isolation of individual minicircles by a transposon-insertion based

approach (see material and method 5.2.4.12). An additional signal was marked by red stars,

representing the gDNA was detectable in all lanes (Fig. 3-1).

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23

Fig. 3-1: The isolated minicircles of A. carterae CCAM0512.

M: 100 bps DNA ladder H3 RTU marker. Lane 1 and 2: Isolated minicircles. Lane 1 and 2 contain a signal in the range of

about 2000 bps. These are the isolated minicircles. The gDNA of A. carterae CCAM0512 was marked by red stars. The gel of

red box area was retrieved and used for the isolation of individual minicircles via a transposon-insertion based approach.

3.1.2 Analysis of individual minicircles

By using a transposon-insertion based approach and several times electroporations (see materials

and methods 5.2.4.12) it was found that twenty two colonies grew on lysogeny broth (LB) agar plate

1 containing kanamycin antibiotic (Table 3-1) and hundreds of thousands of colonies grew on plate 2

and 3. Subsequently, about 150 colonies were picked out and cultured in liquid LB medium

containing kanamycin antibiotic for plasmid preparation (see materials and methods 5.2.4.1). Based

on the digestions by enzymes and analysis on the agarose gel 107 potential positive minicircles were

sequenced via transposon sequencing forward and reverse primers (see supplements 7.3). The 107

potential positive minicircles are named Juan 1 to Juan 107, shortly J1 - J107. Finally, it was found

that 18 out of 89 plasmids (20.2%) are gene-containing minicircles. 71 out of 89 plasmids (79.8%) are

empty minicircles. The number of empty minicircles is about four times the number of gene-

containing minicircles. While the remainder 18 plasmids are false positive colonies. The more detail

will be shown on the next texts.

Table 3-1: Statistic analysis of isolated individual minicircles.

(Plate 1, 2 and 3 are three different transposon insertions and electroporations.)

Plate Colonies on plate

Sequencing number

name False positive minicircles

Gene containing minicircles

Empty minicircles

1 22 10 J1-J6, J21-J22, J70, J71

2 2 6

2 Hundreds of 27 J7-J17, J23-J33, J34-J37, J72

4 8 15

3 Hundreds of 70 J18-J20, J38-J69, J72-J107

12 8 50

Total number

107 18 18(20.2%) 71(79.8%)

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3.1.2.1 The overview of all individual minicircles

Within this 18 gene-containing minicircles, it was found that 12 out of 18 minicircles belong to

different minicircle molecules. The 12 minicircles include three different psbA-containing minicircle

molecules, two different petB/atpA-containing minicircle molecules, two different 23S rRNA-

containing minicircle molecules and one psaB-, psbC-, psbD/E/I-, petD- and atpB-containing

minicircle molecule (Table. 3-2). 11 different genes were identified on these individual minicircles of

2333 bps (J2) – 2664 bps (J33) length from A. carterae CCAM0512 (Table. 3-2). The average length of

all these gene-containing minicircles is 2474 bps. All genes identified represent the core components

of the chloroplast genome of all other photosynthetic organisms, as the encoded subunits of the

complexes (photosystem I and photosystem II, the ATP synthase and the cytb6/f complex) are

involved in the light reactions of photosynthesis as well as 23S rRNA (Table. 3-2). As reported from

minicircles of other species (Hiller 2001, Nisbet, Koumandou et al. 2004), two minicircles containing

more than one gene were isolated, namely J29 (including psbD, psbE and psbI genes) and J37

(including petB and atpA genes). It was also found that these genes on the same minicircle are

separated by only about 100 bps to 600 bps nucleotide sequences. However, in conventional plastid

genome these genes are located much far from each other (Ohyama, Fukuzawa et al. 1986,

Shinozaki, Ohme et al. 1986). In contrast to minicircles from other species (Barbrook and Howe 2000,

Hiller 2001, Zhang, Cavalier-Smith et al. 2002), all genes encoded on minicircles in A. carterae

CCAM0512 have the same start codon (ATG), while TAG, TGA or TAA are used as stop codon in

minicircle genes (Table 3-2).

Table 3-2: Properties of minicircles containing chloroplast genes in A.carterae CCAM0512.

(Minicircles’ number: the number of isolated minicircles; Individual minicircles’ number: the number of different minicircle

molecules; PS I: Photosystem I; PS II: Photosystem II; bps: base pairs)

Circle’ name

Minicircles’ number

Individual minicircles’ number

Coding gene

Minicircle length (bps)

Gene length (bps)

Start codon

Stop codon

Product

J12 1 1 psaB 2469 1875 ATG TGA CP47 PS I

J2 5 3 psbA 2333 1023 ATG TAG D1 PS II

J11 5 1 psbC 2343 1251 ATG TAA CP43 PS II

J29 1 1 psbD/E/I 2354 1068/243/108

ATG TAA D2/Cytochrome b599 alpha/ PS II

J37 2 2 petB/atpA 2596 660/1041 ATG/ ATG

TAA/ TGA

Cytochrome b6/ alpha-subunit ATP synthase

J30 1 1 petD 2481 567 ATG TAA Subunit IV ATP synthase

J28 1 1 atpB 2555 1662 ATG TAG Beta-subunit ATP synthase

J33 2 2 23S rRNA 2664 _ _ _ 23S rRNA

Total number

18 12

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25

In addition to minicircles with coding genes, 71 empty minicircles have been isolated (Table 3-3). Of

the total 71 empty minicircles, 26 (36.6%) belong to different minicircle molecules. The 26 different

molecules were divided into six groups (Table 3-3). For example, the group of J8 empty minicircle

contains three different minicircle molecules in 17 minicircles. These empty minicircles include

different numbers of open reading frames (ORFs) for which no homology could be detected.

Minicircle J9 has nine ORFs>150 bps compared with only two ORFs>150 bps on minicircle J36. Their

lengths are different ranging from minicircle J22 (1449 bps) to minicircle J24 (2493 bps) and

sequences are not conserved. The average length of these empty minicircles is 1986 bps. These ORFs

without known function may be unique to dinoflagellates. It might also be possible that they have

specific functions for minicircles.

Table 3-3: Properties of empty minicircles in A.carterae CCAM0512.

(Minicircles’ number: the number of the isolated minicircles; Individual minicircles’ number: the number of different

minicircle molecules; ORF: open reading frame; bps: base pairs)

Name/Type Minicircles’ number

Individual minicircles’ number

Minicircle length (bps) ORF>150 bps Largest ORF(bps)

J8 17 3 1853 4 249

J9 21 7 1892 9 243

J13 9 6 2209 3 492

J22 1 1 1449 3 195

J24 19 6 2493 6 264

J36 4 3 2020 2 198

Total number 71 26 27

As shown in Table 3-3, these empty minicircles contain several open reading frames with larger than

150 bps without known homology. The lengths of the largest open reading frames in these empty

minicircles are different from 195 bps to 492 bps. In order to detect the relationship between the

ORFs > 150 bps of gene-containing minicircles and empty minicircles the comparison was conducted.

It was found that empty minicircle J9 and atpB-containing minicircle have a completely identical

open reading frame and a partially identical open reading frame (Table 3-4). At the same time, it was

shown that empty minicircles J13, J22 and J36 have an identical open reading frame, empty

minicircles J22 and J24 have an identical open reading frame.

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Table 3-4: Identical open reading frames in A. carterae CCAM0512.

(J28 is atpB containing minicircle, the rest of minicircles including J9, J13, J22, J24 and J36 are empty minicircles. Identical

open reading frames: the minicircles contain the identical open reading frames or partially identical open reading frame.

The potential products of the open reading frames are without any known homology. Only the open reading frames > 150

bps were analyzed here. )

Minicircles Identical open reading frames

J9,J28 MIYFYLVQCNIRDFETQRGFPPPPCRSTLEDPRVPSSNSSQYARTPEKIHR.

J9,J28 ….........LSLIHISTIIDEFSRVFSHIGSNSSSVPGDPLESTCRGGGESHVVSQNL.

J13,J22,J36 MSPQRSIAPFQVICLSAPLPSIEGLSLSFHQLSLHSFVYSLVEILTSRHI.

J22,J24 MSHDIITTTPNPLSFIGGGLIKVKSLWPMRGAIHQLQYLVHSPSYRRSSEH.

For the purpose of distinguishing the difference on the nucleotide sequences of gene-containing

minicircles and empty minicircles the GC contents of these minicircles were compared (Fig. 3-2). It

was shown that the GC contents of the overall minicircles generally appear to be lower than the GC

contents of the coding regions, but higher than the GC content of the non-coding regions except the

same GC content in petB-containing minicircle. A special case is that in psbE and psbI genes-

containing minicircle the overall minicircle presents to be more GC-rich than the coding regions and

the non-coding region, the non-coding region shows higher GC content than the coding regions.

Except the GC content of psbA-containing minicircle is 45%, which is the same as or lower than the

GC content of the overall empty minicircles, all the other gene-containing minicircles present to be

more GC-rich than the empty minicircles. It was also presented that the GC contents of the overall

minicircles, the coding regions and the non-coding regions are lower than the AT content.

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Fig. 3-2: GC content for minicircles of A. carterae CCAM0512.

The figure shows that the GC content of the overall minicircles, the coding regions and the non-coding regions in genes-

containing minicircles and empty minicircles. It was shown that the overall GC contents of the minicircles are generally

lower than the GC content of the coding regions, but higher than the GC content of the non-coding regions except the

same GC content in petD gene minicircle. However, the overall psbE and psbI-containing minicircles appear to be more GC-

rich than the coding regions and the non-coding regions. Except the psbA-containing minicircle all the other gene-

containing minicircles present to be more GC-rich than the empty minicircles. The more detailed description see text. GC

content is calculated on the website http://emboss.bioinformatics.nl/.

3.1.2.2 The core regions of minicircles in A. carterae CCAM0512

Fig. 3-3: Alignment of the core regions of fourteen minicircles of A. carterae CCAM0512.

It was shown that all fourteen minicircles have a highly conserved sequence about 60 bps called core region, and a 12 bps

conserved sequence called core out region. Only the conserved regions were shown in this figure. Consensus regions of all

fourteen minicircles of A. carterae CCAM0512 were aligned by using ClustalX 2.1.

In order to compare the non-coding regions of all minicircles the sequences of consensus minicircles

were aligned. It was shown that there is a highly conserved core sequence of 60 bps, called core

region. At the same time, there is a 12 bps conserved sequence called core out region (Fig. 3-3).

http://emboss.bioinformatics.nl/

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28

Based on the comparison of the sequences between the core region and the core out region it was

found that the nucluotides of minicircle J8, J11, J12, J28, J29, J30, J33 and J37 are the same, but

different from that of the remainder six minicircles. To predict the function of the core region

different algorithms were used to predict a putative promoter sequence, whereby the sequence

“CACCAGCTTCAAAAAATGCCGGTCAATCCATAGGAGTGAGAAAATCACAG/TGATGAGA” was predicted

to have a putative function for initial transcription, which has to be confirmed in the future.

3.1.2.3 Transcription and RNA editing analyses of individual minicircles

RNA-editing can be commonly observed in land plants, although the frequency varies in different

lineages. In the algae, the RNA editing remains undetectable until now, aside from the editing that

occurred in the dinoflagellates Ceratium horridum and Heterocapsa triquetra (Zauner, Greilinger et

al. 2004, Dang and Green 2009). In order to study the RNA editing on minicircles of A. carterae

CCAM0512 mRNA derived sequences were compared with genomic sequences of the psbA gene. It

was shown that no RNA editing was observed in the psbA gene. The observed RNA products support

the previous study that the transcription of coding genes on minicircles (Barbrook and Howe 2000,

Barbrook, Symington et al. 2001, Zhang, Cavalier-Smith et al. 2002, Nisbet, Hiller et al. 2008).

Fig. 3-4. Transcription of ORFs of three empty minicircles.

It was indicated that the three ORFs were transcribed in A. carterae CCAM0512. The figure showed the results of RT-PCR

by using the primers designed to amplify the regions of three largest ORFs of different empty minicircles (J9, J13 and J24).

Lane 1, lane 3 and lane 5: control PCR without reverse transcriptase added. Lane 2, lane 4 and lane 6: The transcriptional

products of ORFs on empty minicicle J9, J13 and J24. The lengths are 243 bps, 492 bps and 264 bps, respectively. M: DNA

markers.

It was already shown that transcription occurs over a large part of the empty minicircle including the

core region (Nisbet, Hiller et al. 2008). Here, the reverse transcriptase-PCR (RT-PCR) was carried out

in order to analyse the transcription on the largest ORFs of empty minicircles (J9, J13 and J24, see

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table 3-2). Products were observed for all three ORFs (Fig. 3-4) and comfirmed by sequencing, which

indicated that these ORFs of empty minicircles might be transcribed.

3.1.3 Evolution analyses of four A. carterae strains’ minicircles

Until now, minicircles have been identified in four different Amphidinium carterae strains, including

A. carterae CCAM0512 presented here and A. carterae CS-21, A. carterae CCAP1102/6 and A.

carterae CCMP1314. The protein-coding genes identified on minicircles of the different A. carterae

strains are listed in table 3-5 (Barbrook and Howe 2000, Hiller 2001, Zhang, Cavalier-Smith et al.

2002, Barbrook, Santucci et al. 2006). All these genes are important for the function of plastid.

However, the reported genes on minicircles remain very limited. To compare the minicircles’

relationship of these four A. carterae strains phylogenetic analysis were performed for the coding

sequences and non-coding sequences in this chapter.

Table 3-5: Reported minicircular sequences in four A.carterae strains.

(LSU: large subunit; SSU: small subunit; rRNA: ribosome RNA)

strains atpA atpB petB petD psaA psaB psbA psbB psbC psbD psbE psbI

LSU-rRNA

SSU-rRNA

A. carterae CCAM0512

✓a ✓ ✓a ✓

✓ ✓ ✓ ✓ ✓b ✓b ✓b ✓

A. carterae CCAP1102/6

✓a ✓ ✓a ✓ ✓ ✓ ✓ ✓ ✓ ✓b ✓b ✓b ✓ ✓

A. carterae CS21

✓a ✓ ✓a ✓ ✓ ✓ ✓ ✓ ✓ ✓b ✓b ✓b ✓ ✓

A. carterae CCMP1314

✓

✓

Genes located on the same minicircles are indicated by superscript lettering a and b. Minicircles in A. carterae CCAP 1102/6,

A. carterae CS21 and A. carterae CCMP1314 see references (Barbrook and Howe 2000, Barbrook, Symington et al. 2001,

Hiller 2001, Zhang, Cavalier-Smith et al. 2002, Nisbet, Koumandou et al. 2004, Barbrook, Santucci et al. 2006). PsbB

mincircle was isolated by Groche C. (Grosche 2012).

3.1.3.1 Overall genome characteristics and open reading frames

Based on the comparison of the GC content of the psbA gene-containing minicircles, it was shown

that the GC contents of the coding regions are the same in these four A. carterae strains and higher

than the GC content of the overall minicircles, the non-coding regions and core regions (Fig. 3-5a). It

generally appears to be more GC-rich in A. carterae CCMP1314 than the other A. carterae strains.

The GC contents of the overall minicircles in A. carterae CCAP1102/6 and A. carterae CCMP1314 are

higher than that of A. carterae CCAM0512 and A. carterae CS21. The GC content of A. carterae CS21

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30

non-coding regions is 43%, which is lower than that in the other three species. In the core regions,

the GC content increases progressively from A. carterae CCAM0512, A. carterae CCAP1102/6, A.

carterae CS21 to A. carterae CCMP1314 (Fig. 3-5a). For the 23S rRNA containing minicircles the GC

contents of the overall minicircles are the same, as shown in Fig. 3-5b.

Fig. 3-5: GC content of psbA and 23S rRNA-containing minicircles in four A. carterae strains.

The figure shows that the GC contents of the overall minicircles, the coding regions, the non-coding regions and the core

regions in four A. carterae strains. a: GC content for psbA-containing minicircles. It was shown that the GC contents of the

coding regions are nearly the same and are higher than the GC contents of the overall minicircles, the non-coding regions

and the core regions. The more detailed description see text. b: GC content for 23S rRNA containing minicircles. It was

shown that the GC contents of the overall minicircles are the same on these four A. carterae strains. GC content is

calculated on the website http://emboss.bioinformatics.nl/.

Like shown in table 3-4, several identical open reading frames (the lengths > 150 bps) of the gene-

containing minicircles and empty minicircles were found in A. carterae CCAM0512, they do not have

known homology. The open reading frames (the lengths > 150 bps) were also compared in four A.

carterae strains. It was shown that 7 out of 27 pairs of open reading frames have a high query cover,

low e-value and high identity (Table 3-6). Among these three pairs of open reading frames have a

100% query cover, very low e-value and considerable high identity (83%, 98% and 96%) (marked by

red).

http://emboss.bioinformatics.nl/

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31

Table 3-6: The open reading frames of empty minicircles in A. carterae strains.

(The name is consist of the empty minicircle name and open reading frame number in A. carterae CCAM0512---accession

number and open reading frame number in A. carterae CCAP1102/6 and CS21 strains. Accession numbers of sequences in

A. carterae CCAP1102/6: [AJ582641] [AF401630]. Accession numbers of sequences in A. carterae CS21: [AJ307015]

[DQ507216] [AJ318067]. The sequences of the open reading frames see supplements 7.1. The comparison was performed

on the website http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome )

Name Max score total score query cover e-value identity

J8 ORF2---AJ307015 ORF1 67.4 90.9 70% 5e-16 100%

J8 ORF3---AJ582641 ORF1 38.5 38.5 29% 3e-06 100%

J13 ORF2---DQ507216 ORF1 251 251 100% 2e-80 83%

J22 ORF1---AF401630 ORF1 103 103 100% 5e-29 98%

J24 ORF2---AF401630 ORF2 65.5 65.5 61% 2e-15 91%

J24 ORF2---AJ318067 ORF1 56.6 56.6 56% 2e-12 88%

J24 ORF6---AJ318067 ORF2 112 112 100% 7e-32 96%

3.1.3.2 Phylogenetic analysis of psbA genes for 15 minicircles of dinoflagellates

Fig. 3-6. Phylogenetic tree analysis based on amino acid sequences encoded by psbA gene from 15

minicircles of dinoflagellates.

It was shown that the four A. carterae strains form a tightly clade with a strong statistical support (bootstrap support

values: 99%, marked by red box). It was indicated that the coding regions of psbA gene for the four A. carterae strains are

almost identical. All the other species was found to form an independent clade from the clade consisting of A. carterae and

A. massartii. The phylogenetic tree was built by maximum likelihood analysis using Mega 6.0. The tree with the highest log

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

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32

likelihood (-1923.2228) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions

per site. The analysis involved 15 amino acid sequences. All positions containing gaps and missing data were eliminated.

There were a total of 336 positions in the final dataset. The bootstrap consensus tree inferred from 1000 replicates. Scale

bar represents 0.02 expected substitutions per site in the aligned regions.

Phylogenetic analysis of PsbA encoded on minicircles from different peridinin-containing

dinoflagellate species showed that A. carterae CCAM0512 forms an independent and tight clade

with the other three different A. carterae strains, thus indicating that the four strains are closely

related to each other with strong statistical support (bootstrap support values: 99%) (Fig. 3-6). The

psbA coding sequences of these four A. carterae strains are almost identical. At the same time, these

results could be confirmed by the phylogenetic analysis for amino acid sequences of other proteins

encoded on minicircles (data not shown). As shown in Fig. 3-6, Heterocapsa and Symbiodinium

species form an independent clade, respectively separated from the clade containing A. carterae.

3.1.3.3 Alignment analysis of core regions from four A. carterae strains

Fig. 3-7. Alignment of the core region of different A. carterae strains’ minicircles.

It was shown that the core regions are highly diverse and unrelated between these four strains. The core regions of four A.

carterae strains (A. carterae CCMP1314, A. carterae CCAP1102/6, A. carterae CCAM0512 and A. carterae CS21) were

compared by using ClustalX 2.1.

It was already shown that the psbA coding sequences encoded on minicircles of four A. carterae

strains are highly identical and these four strains form a tightly sister-relationship in a clade.

Therefore, it is essential to compare the core regions of these four strains. Surprisingly, the core

regions of strain CCAM0512 and strain CS-21 are highly diverse in comparison to each other and to

the core regions of the strains CCMP1314 and CCAP1102/6 (Fig. 3-7).

The coding sequences and core regions of the four A. carterae strains have already been compared.

Finally the remainder non-coding regions of psbA minicircles were also compared for the four A.

carterae strains (Fig. 3-8). For psbA there is a 13 bps conserved sequence downstream the stop

codon, upstream the start codon there are four longer conserved regions. However, the sequences

are highly variable in the rest of the non-coding regions. It was shown that the core regions are

located in a more variable region. To predict the function of the conserved sequence upstream the

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33

start codon of psbA different algorithms were used to predict a putative promoter sequence,

whereby a conserved sequence of 50 bps might have a function for the initial transcription, which

has to be confirmed in the future.

Fig. 3-8. Alignment of the non-coding regions of four different A. carterae strains’ minicircles.

“TAG” is the stop codon of the psbA gene, while “ATG” is the start codon. The core regions of four A. carterae strains’

minicircles were truncated and marked by red triangles. It was shown that the core regions are located in a more variable

region. Upstream the start codon and downstream the stop codon of psbA gene (marked by the red boxes) of the non-

coding sequences are conserved. The putative promoter region was marked by blue box. The non-coding regions without

core regions of four A. carterae strains’ psbA minicircles were compared by using ClustalX 2.1.

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34

According to these analyses, the sequences of the A.carterae strain CCAP1102/6 and the A.carterae

strain CCMP1314 are almost 99% identical therefore it was speculated that they are probably the

same strains.

3.1.3.4 Phylogenetic analysis of partial LSU/SSU rDNA

With the purpose of comparing the evolutionary relationship of these four A. carterae strains further

phylogenetic analyses based upon two nuclear genes (partial LSU rDNA and SSU rDNA) were carried

out. The A. carterae strains formed clades that were unambiguously separated from some other

Amphidinium species (Fig. 3-9 and 3-10). The tree obtained for LSU rDNA sequences showed that all

A. carterae species formed a tightly sister relationship within the same clade (Fig. 3-9). This clade

was divided into three subclades, containing the strain A. carterae CCMP1748 and another two

larger clades (clade 1 and clade 2). Additionally, it was shown that the strains of A. carterae CS-21

and A. carterae CCAM0512 are in the same clade, forming a tight neighbor relationship with strong

statistical support (bootstrap support values: 100%) (Fig. 3-9). The phylogenetic position of A.

carterae species based upon its SSU rDNA sequences was shown in Fig. 3-10. All the A. carterae

species were grouped in the same clade. The species A. carterae CCMP1314, A. carterae CCAP1102

and A. carterae CCAM0512 were found to form a sister relationship, which was confirmed by the

high bootstrap support values.

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35

Fig. 3-9. Phylogenetic tree analysis based on LSU rDNA sequences covering domains D1-D6.

It was shown that all the A. carterae species form an independent clade (marked by red arrow at the node). This A.

carterae clade was divided into three subclades, clade 1, clade2 and A. carterae CCMP1748. A. carterae CS-21 and A.

carterae CCAM0512 were contained in the clade1 and form the closest relationship (marked by the red box). The

corresponding GenBank accession numbers of sequences are after the each taxa. The phylogenetic tree was made using

Maximum Likelihood method based on the large subunit rDNA sequences covering domain 1 to domain 6. The tree with

the highest log likelihood (-5458.4502) is shown. Bootstrap values (>50%) are given at each node. Evolutionary analyses

were conducted in MEGA6 (Tamura, Stecher et al. 2013).

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Fig. 3-10. Phylogenetic tree analysis based on SSU rDNA sequences.

All the A. carterae species form an independent clade together with A. operculatum CCMP1342, A. cf. rhynchocephalum

UTEX and A. massartii CCCM 439 (marked by red arrow at the node). A. carterae CCAM0512, A. carterae CCMP1314 as well

as A. carterae CCAP1102 form a nearest phylogenetic neighbours (marked by the red box). The corresponding GenBank

accession numbers of sequences are after the each taxa. The phylogenetic tree was carried out as described in Fig. 3-9.

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3.2 The endomembrane system (ES) in Phaeodactylum tricornutum

The localization of proteins on a subcellular level is a very important technique for cell biology

research. In recent years, the most common used method for protein localization is to fuse the

proteins of interest to a reporter, whereby examples include the enhanced green fluorescent protein

(eGFP) and monomeric red fluorescent protein (mRFP) and express these fusion proteins in the cells.

The already known marker proteins in diatoms are not enough for studying the mechanisms of

vacuolar protein sorting and transport, therefore establishing further subcellular-specific marker

proteins in endomembrane system is the aim of this work.

In this project, to study subcellular-specific localization of interest proteins eGFP- and mRFP-fusion

proteins were generated and the confocal laser scanning microscope was used to identify the

subcellular localization of proteins in vivo in P. tricornutum.

3.2.1 Identification of marker proteins

Marker proteins with known localization are not enough for the investigation of the mechanisms of

vacuolar protein sorting and transport in P. tricornutum. In order to identify more marker proteins

proteins with a known localization in plants were collected from the literature. All homologous

genes were extracted from the P. tricornutum genome database.

In order to correct the gene models retrieved from the database, the predicted gene model

sequences have been compared with expressed sequence tags (EST). Simultaneously, the start

codons upstream the predicted proteins were screened on the genome browser. If it was necessary,

the gene models were corrected. If there was no EST available, the corresponding cDNAs were

amplified and sequenced. In addition, for each protein the presence of targeting signals and

transmembrane domains (TMDs) were predicted with different servers (see material and methods

chapter). Subsequently, putative marker proteins were selected in order to analyze their in vivo-

localization. An overview of all proteins can be found in the following chapters.

3.2.2 Tonoplast intrinsic proteins (Tips)

The first identified proteins are tonoplast intrinsic proteins (Tips). Tips are one of the aquaporins

family groups. Aquaporins are channel proteins belong to the major intrinsic proteins (MIPs)

superfamily. The tonoplast intrinsic proteins form transmembrane channels that selectively mediate

the transport of water, gases and small neutral solutes such as glycerol and urea (Gattolin, Sorieul et

al. 2009). This protein family was found in a variety of plants, animals and bacteria and have a high

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sequence similarity to each other (Johnson and Chrispeels 1992, Jauh, Phillips et al. 1999, Liu,

Ludewig et al. 2003, Moriyasu, Hattori et al. 2003, Zardoya 2005). Tips were widely used as markers

for lytic vacuole and protein storage vacuole in higher plants (Frigerio, Hinz et al. 2008, Gattolin,

Sorieul et al. 2009). The localization and function of several Tip isoforms (including three gamma-Tip

(Tip1), three delta-Tip (Tip2), the seed specific alpha- and beta-Tip (Tip3:1 and Tip3:2), one epsilon-

Tip (Tip4:1) and one zeta-Tip (Tip5:1)) have already been well-studied or predicted in plant species,

but it remains unknown about the localization of Tip proteins in diatoms.

Finally, five Tip proteins (Tip1-5) homologous to Tip proteins of A. thaliana were identified and

selected for in vivo-localization studies in P. tricornutum (Table 3-7). All these Tip proteins have six

putative transmembrane domains (called TMD1-TMD6). Based on the observation on the amino acid

sequence of the five Tip proteins it was shown that each Tip protein has one (in case of Tip3 and

Tip4) or two (in case of Tip1, Tip2 and Tip5) conserved Asn-Pro-Ala (NPA) motifs between two TMDs

to form extra-cytosolic loop helices. The prediction of targeting signals showed that only Tip4 has a

signal peptide. All the other Tips also comprise the specific domain structure, but they are lack of a

predictable signal peptide.

Table 3-7: Predicted subcellular localized Tonoplast intrinsic proteins in P. tricornutum.

(Pt: P. tricornutum, P: plastid (envelope), PPM: periplastidal membrane, EM: endosomal membrane, SP: signal peptide;

TMD: transmembrane domain; loc: localization)

protein family name BLAST hits/specific function Pt ID SP TMD Predicted-loc

eGFP-loc

Tonoplast intrinsic protein (TIP)

Tip1 aquaporin 31553 - 6 EM Plasma membrane

Tip2 glycerol uptake facilitator - - 6 EM Vacuole

Tip3 glycerol uptake facilitator 20755 - 6 EM cER/nuclear envelope

Tip4 glycerol uptake facilitator 19409 + 6 P/PPM PPM

Tip5 aquaporin 43157 - 6 PPM cER/nuclear envelope

3.2.2.1 In vivo-localization of Tip1

In order to study the subcellular localization of Tip1, eGFP was attached to the C-terminus of the

protein and were expressed in P. tricornutum. The fluorescence pattern of the Tip1-eGFP fusion

protein surrounded the cell. Therefore it was speculated that Tip1 localized to the cytoplasmic

membrane (Fig. 3-11a). However, besides the plasma membrane, one or several dots inside the cells

could be observed in some clones (Fig. 3-11b). This intracellular fluorescence might stem from

endocytosed compartments. To test this, co-staining with FM4-64 was carried out. This dye is used

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visualize the plasma membrane and endomembrane system-dependent internalization processes.

The co-localization of Tip1-eGFP with FM4-64 indicates that the internal Tip1-eGFP fluorescence

belongs to the endosomal system (Fig. 3-11c). Based upon the prediction, Tip1 protein is a

membrane protein. In order to confirm this carbonate extraction was performed. As shown in Fig. 3-

11d, the Tip1-eGFP fusion protein was inserted into the membrane, although a weak signal could be

detected in the fraction of soluble proteins.

Fig. 3-11: In vivo localization of Tip1-eGFP.

a: The Tip1-eGFP fusion protein showed a plasma membrane fluorescence; b: An additional dot-like fluorescence could be

observed in some clones. c: The intracellular dots of Tip1-eGFP overlapped with FM4-64. d: Carbonate extraction of Tip1-

eGFP. Although a weak signal could be observed in the fraction of soluble, it was shown that Tip1 is membrane protein as

the signal was very strong in fraction of integral membrane. The thylakoid membrane protein PsbD (25 kDa) and the

stromal protein RbcL (55 kDa) were used as makers for the fraction of soluble and integral membrane proteins. The

expected molecular weight of Tip1-eGFP protein is 60 kDa. For a detailed description see text. TL (gray): transmitted light;

PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent protein; FM4-64 (yellow): a dye used to

visualize the plasma membrane and endomembrane system-dependent internalization processes, stained for 25min;

overlay: the overlay of PAF, GFP and FM4-64.


In plant cells the vacuole is the largest subcellular compartment. Until now no vacuolar proteins

have been reported in diatoms. In order to visualize this single membrane surrounded structure,

Tip2 was selected for in vivo localization studies in P. tricornutum.

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After in vivo localization the Tip2-eGFP fusion protein was targeted to the vacuolar-like structure (Fig.

3-12a). By carbonate extraction, it was shown that Tip2-eGFP is an integral membrane protein (Fig.3-

12b).


a: Tip2-eGFP fusion protein was expressed. The fusion protein was targeted to a vacuolar-like structure. b: Carbonate

extraction of the Tip2-eGFP fusion protein. It was shown that Tip2 is a membrane protein. The thylakoid membrane

protein PsbD (25 kDa) and the stromal protein RbcL (55 kDa) were used as makers for the fraction of soluble and integral

membrane proteins. The expected molecular weight of Tip2-eGFP is 55 kDa. For a detailed description see text. TL:

transmitted light; PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent protein.

3.2.2.3 In vivo-localization of Tip3 and Tip5

Co-evolution of the host cell and the endosymbiont gave rise to the establishment of the secondary

or complex platids. The secondary or complex plastids are surrounded by four membranes (the

outermost membrane, cER; the second outermost membrane, PPM; the outer and inner envelope

membranes). The membranes of the host ER, the nuclear envelope and the outermost plastidal

membrane (cER membrane) are connected to each other. To better study the ER function, it is

necessary to have additional marker proteins. Here, two homologous tonoplast intrinsic proteins

Tip3 and Tip5 were selected for localization studies in P. tricornutum.

As shown in Fig.3-13a and b, the fluorescence of Tip3-eGFP and Tip5-eGFP could be detected in the

cER-membrane and the nuclear envelope. However, in most cases fluorescence could also be

observed in additional structures, which needs to be confirmed in the future. By the carbonate

extraction the signal was mainly observed in the fraction of integral membrane (Fig. 3-13c and d). It

indicated that Tip3 and Tip5 were targeted to the membrane, which is consistent with the prediction

in silico.

To verify the localization of Tip3-eGFP immunogold labelling and electron microscopy was

performed. As shown in Fig. 3-13e, the immunogold particles predominantly label the outermost

membrane of the complex plastid (cER membrane) and the nuclear envelope. At the same time,

several additional immunogold particles could be detected (marked by red arrow), which might hint

that Tip3-eGFP was also located in the host ER.

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Fig. 3-13: In vivo localization of Tip3- and Tip5-eGFP.

a-b: Tip3- and Tip5-eGFP fusion proteins were expressed under the control of a nitrate inducible promoter. The

fluorescence of Tip3-eGFP and Tip5-eGFP fusion proteins were targeted to the cER membrane and nuclear envelope. In

most cases additional fluorescence extended from the cER membrane and nuclear envelope was also observed. c and d:

Carbonate extraction of Tip3- and Tip5- eGFP. It indicated that Tip3 and Tip5 are integral membrane proteins. The

thylakoid membrane protein PsbD (25 kDa) and the stromal protein RbcL (55 kDa) were used as makers for the fraction of

soluble and integral membrane proteins. The expected molecular weight of Tip3/ Tip5-eGFP proteins are 56 kDa and 60

kDa, respectively. e: Immunoelectron microscopic analyses on ultra-thin cuts of P. tricornutum, indicating that Tip3 was

significantly targeted into the outermost membrane of the complex plastid (cER membrane) (marked by black arrows).

Immunogold labeling was partially accumulated in the nuclear envelope and might be on the host ER (marked by red

arrows). For a detailed description see text. TL: transmitted light; PAF (red): plastid autofluorescence; eGFP (green):

enhanced green fluorescent protein.

In order to verify the subcellular localization of the Tip5-eGFP, co-localization was performed

between Tip5-eGFP and DAPI. Partial fluorescence of Tip5 surrounded the DAPI staining, which

further confirmed the fluorescence of Tip5 was targeted to the nuclear envelope (Fig. 3-14a). Co-

expression of Tip5-eGFP and Tip3-mRFP was carried out and verified the co-localization of these

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proteins (Fig. 3-14b). However, a strong dot-like fluorescence could be detected on the mRFP

channel in some cases (data not shown), which might result from the overexpression of the mRFP

fusion protein. One of the important components of the host ERAD machinery hDer1 has already

been identified to enrich in the cER membrane, nuclear envelope and the host ER (Hempel,

Bullmann et al. 2009). The co-expression of hDer1-eGFP and Tip3-mRFP indicated that these

proteins have the same subcellular localization, but the fluorescence was distributed differently (Fig.

3-14c). To combine all the results Tip3 and Tip5 might localize in the membrane of cER, nuclear

envelope and the host ER.

Fig. 3-14: In vivo co-staining and co-expression analyses of Tip3 and Tip5 proteins.

a: Co-localization of Tip5-eGFP with DAPI showed that partial fluorescence of the Tip5-eGFP was targeted to the nuclear

envelope. b: Co-expression of Tip3-mRFP and Tip5-eGFP showed that these two proteins colocalize in the cER membrane

and nuclear envelope. An additional fluorescence extended from the cER membrane and nuclear envelope was also

observed. c: Co-expression of hDer1-eGFP and Tip3-mRFP showed that these two proteins are targeted to the same

compartments (cER membrane, nuclear envelope and host ER), but the fluorescence was distributed differently. TL:

transmitted light; PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent protein; DAPI (blue): 4′,6-

Diamidin-2-phenylindol; mRFP (Magenta): monomeric red fluorescent protein; overlay: the overlay of PAF, eGFP and mRFP.


Likewise, the subcellular localization of Tip4 was analyzed in P. tricornutum. It was known that the

complex plastid is surrounded by four membranes. The periplastidal membrane (PPM) is the second

outermost membrane of the complex plastid which is located between the periplastidal

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compartment (PPC) and the chloroplast ER (cER) space. Previous study showed that N-terminal

bipartite targeting sequences (BTS) are specific signals for importing proteins into the complex

plastid (PPC or PPM) (Gruber, Vugrinec et al. 2007). Interestingly, Tip4 is the only Tip which

possesses a potential signal peptide.

Subcellular localization studies of Tip4-eGFP showed that the fluorescence pattern results in a

classical “blob-like” structure known to represent a PPC/PPM localization, as shown in Fig. 3-15a. To

further confirm this localization, the self-assembly GFP assay was performed. It was shown that the

C-terminus of Tip4 was located in the PPC, as fluorescence signals could be observed upon co-

expression of Tip4-S11 with a GFPS1-10 fused to a PPC marker but not fused to an ER marker (Fig. 3-

15b). Compared with the subcellular localization of sDer1-2 (Gruber, Vugrinec et al. 2007, Hempel,

Bullmann et al. 2009) Tip4 does not have an aromatic amino acid at the +1 position of the predicted

transit peptide for plastid import. The PPM localization of Tip4 was concluded.


a: Subcellular localization of Tip4-eGFP indicated that it was targeted a characteristic PPM/PPC-like compartments. b: The

self-assembly GFP assay was used to confirm the localization of Tip4. GFP-11 was fused to the C-terminus of full-length of

Tip4 and co-expressed together with Hsp70BTS-S1-10 (a PPC marker) and PDI-S1-10 (an ER marker), respectively. Only after

co-expressed of Tip4-S11 with the PPC marker (lower pannel) but not after co-expression with the ER marker (upper

pannel) a GFP signal was detectable. Self-assembly GFP assay results showed that the C-terminus of Tip4 is located on the

PPC. TL: transmitted light; PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent protein.

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3.2.3 In vivo localization of Golgi proteins

The Golgi apparatus consists of three main networks (the cis-Golgi network, the medial Golgi and the

trans-Golgi network). In order to identify marker proteins for the slightly different Golgi networks, a

large-scale search for proteins homologous to known Golgi localization proteins was conducted in

the genome of P. tricornutum. Finally three proteins homologous to known enzymes involved in the

N-Glycosylation pathway were selected for in vivo-localization in P. tricornutum (Table. 3-8).

Table 3-8: Predicted subcellular localized Golgi marker proteins in P. tricornutum.

(Pt: P. tricornutum; SP: signal peptidase; TMD: transmembrane domain; loc: localization). The identifications and cloning of

GnT1 and FucT was conducted by Clément Ovide.

name BLAST hits/specific function Pt ID SP TMD Predicted-loc

eGFP-loc

N-acetylglucosaminyltransferase I (GnT1)

catalyse N-glycoproteins transported from ER to Golgi

54844 - 1 Cis-Golgi Cis-Golgi

α-mannosidase I (α-Man I)

enzyme involved in N-glycan biosynthesis; Transfers an alpha-D-mannosyl residue from dolichyl-phosphate D-mannose into membrane lipid-linked oligosaccharide

44425 + 6 Cis-Golgi unknown

β1,2-xylosyltransferase (XylT)

glycosyltransferase involved in N-glycan biosynthetic pathway

45496 + 1 Medial-Golgi

Medial-Golgi

α1,3-fucosyltransferase (FucT)

glycosyltransferase involved in N-glycan biosynthetic pathway

54599 + 1 Trans-Golgi/TGN

Trans-Golgi/TGN

α1,3-Mannosyltransferase (α1,3-Man)

enzyme involved in N-glycan biosynthesis; Transfers an alpha-D-mannosyl residue from dolichyl-phosphate D-mannose into membrane lipid-linked oligosaccharide

22554 + - Medial-Golgi

unknown

The first one GnT1 is known to be localized in the cis-Golgi (Kajiura, Okamoto et al. 2012). The

second protein XylT is a type II membrane proteins that belongs to the glycosyltransferase family 61

and has been confirmed to localize on medial cisternae of the Golgi apparatus (Pagny, Bouissonnie

et al. 2003, Kajiura, Okamoto et al. 2012). The third one FucT is involved in the transfer of alfa-1,3-

linked fucose residues to N-glycans and is expected to be targeted to the TGN (Fitchette‐Lainé,

Gomord et al. 1994, Breton, Mucha et al. 2001). By expressing all three as eGFP fusion proteins a

dot-like structure or a long strip fluorescence pattern was observed (Fig. 3-16a/b/c). By carbonate

extraction it could be shown that XylT is an integral membrane protein, as shown in Fig. 3-16d.

To confirm whether the localizations of these three Golgi marker proteins are slightly different, co-

expression of XylT-mRFP/ GnT1-eGFP and XylT-mRFP/ FucT-eGFP was performed. It was shown that

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45

the eGFP and mRFP fluorescence partially overlapped for both co-expressions (Fig. 3-16e/f). All

these results showed that these three proteins were targeted into different cisternae of the Golgi

apparatus.

Fig. 3-16: In vivo localization of proteins located in the Golgi apparatus.

a-c: The GnT1-, XylT- and FucT-eGFP fusion proteins showed a similar dot-like or long strip-like fluorescence pattern. d:

Carbonate extraction of XylT-eGFP. The thylakoid membrane protein PsbD (25 kDa) and the stromal protein RbcL (55 kDa)

were used as makers for the fraction of soluble and integral membrane proteins. The expected molecular weight of XylT-

eGFP proteins is 83 kDa. e and f: Co-expression of XylT-mRFP/ GnT1-eGFP and XylT-mRFP/ FucT-eGFP showed eGFP and

mRFP fluorescence partially overlapped. g and h: The overlay of XylT-mRFP/ GnT1-eGFP and XylT-mRFP/ FucT-eGFP was

enlarged, respectively. TL: transmitted light; PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent

protein; mRFP (Magenta): monomeric red fluorescent protein; overlay: overlay of PAF, eGFP and mRFP.

3.2.4 In vivo localization of retromer complex

The structure and function of the retromer complex have already been well identified in yeast,

animals and some plants, while the retromer complex in diatoms is still not much known. In

Arabidopsis thaliana, the retromer complex comprises a small subcomplex (three sorting nexins) and

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46

a larger subcomplex (Vps26, Vps29 and Vps35). In order to localize the putative retromer subunits in

P. tricornutum, several proteins homologous to retromer subunits in A. thaliana were analyzed in P.

tricornutum (Table 3-9).

Table 3-9: Predicted subcellular localization of retromer complex proteins in P. tricornutum.

(Pt: P. tricornutum; SP: signal peptidase; TMD: transmembrane domain; loc: localization)

Retromer subunits BLAST hits/specific function Pt ID SP TMD Predicted-loc

eGFP-loc

Vacuolar protein sorting- associated protein 26 (Vps26)

Inform multi-protein complexes, Intracellular protein transport

41962 - - TGN/EE Golgi/EE


Inform multi-protein complexes , Intracellular trafficking, secretion and vesicular transport

17936 - - TGN/EE Golgi/EE


Inform multi-protein complexes that facilitate retrograde transport of lytic vacuolar-targeting receptors back to the trans-Golgi network

43830 + - TGN/EE Golgi/EE

Sorting nexin dimer (SNX1) Membrane coat complex retromer, subunit VPS5/SNX1, sorting nexins, and related PX domain-containing proteins

3137 - - TGN/EE unknown

Homologs of the potential subunits Vps26 and Vps29 were selected for in vivo localization. The

fluorescence patterns of these eGFP fusion proteins were similar and showed a dot or small

diamond-like structure (Fig. 3-17a and b). In order to make sure that they are colocalization as

predicted, the co-expression of Vps26-eGFP and Vps29-mRFP was performed. It was shown that

these the fluorescence pattern overlapped completely (Fig. 3-17c and d). Taking together, it was

speculated that two potential homologs (Vps26 and Vps29) of proteins in A. thaliana might also

locate in one complex in P. tricornutum.

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47

Fig. 3-17: In vivo localization of retromer complex proteins.

a-b: The Vps26- and Vps29-eGFP fusion proteins were expressed in P. tricornutum. It was shown that the fluorescence

patterns are similar, a dot or a small diamond-like structure. c: The co-expression of these two potential retromer complex

subunits showed that the fluorescence pattern overlapped. d: The overlay of Vps26-eGFP/ Vps29-mRFP was enlarged. TL:

transmitted light; PAF (red): plastid autofluorescence; eGFP (green): enhanced green fluorescent protein; mRFP (Magenta):

monomeric red fluorescent protein; overlay: the overlay of PAF, eGFP and mRFP.

In order to distinguish the Golgi marker proteins and retromer co-expression of one retromer

subunit (Vps29) and the medial Golgi marker protein XylT was carried out. Only partial overlapped

fluorescence was observed (Fig. 3-18a and enlarged picture b). However, the co-expression of the

Vps29 together with the trans-Golgi marker protein FucT showed that their fluorescence overlapped

completely (Fig. 3-18c and d). These co-localizations further indicated that the XylT and FucT was

located on different cisternaes of the Golgi apparatus and the retromer subunit was targeted to the

trans-Golgi network.

Fig. 3-18: Co-expression of retromer complex proteins with Golgi markers.

a: Co-expression of the medial Golgi marker XylT and the retromer subunit Vps29 showed that their fluorescence

overlapped partially. b: The overlapped fluorescence of XylT-mRFP with Vps29-eGFP was enlarged. c: Co-expression of the

trans-Golgi network marker FucT and the retromer subunit Vps29 showed the fluorescence overlapped completely. d: The

overlapped fluorescence of FucT-eGFP with Vps29-mRFP was enlarged. TL: transmitted light; PAF (red): plastid

autofluorescence; eGFP (green): enhanced green fluorescent protein; mRFP (Magenta): monomeric red fluorescent protein;

overlay: the overlay of PAF, eGFP and mRFP.

3.2.5 In vivo localization of vacuolar H+-ATPase proteins

Previous studies have already shown that two different vacuoles exist in plant cells, namely the

central lytic vacuole and the protein storage vacuole (Marty 1999, Jin, Kim et al. 2001, Park, Kim et al.

2004). In order to mark these different types of vacuoles, more putative vacuolar candidates were

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analyzed in vivo localization. It has already shown that vacuolar H+-ATPases (VHAs) are localized to

vacuoles and other endosomal membranes, including the ER, Golgi apparatus and vesicles as well as

the plasma membrane (Sze, Schumacher et al. 2002). Therefore proteins homologous to VHAs of A.

thaliana were analyzed in silico in P. tricornutum (Table 3-10).

Table 3-10: Predicted subcellular localization of V-ATPase proteins in P. tricornutum.

(Pt: P. tricornutum; SP: signal peptidase; TMD: trans-membrane domain; PM: plasma membrane; loc: localization)

name BLAST hits/specific function Pt ID SP (TargetP RC)

TMD Predicted-loc eGFP-loc

ATPase1 Vacuolar H+-transporting two-sector ATPase, C subunit

21882 + 4 PM/Vacuole Lytic vacuole?

ATPase2 Vacuolar ATP synthase subunit G2; hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances; proton transport

44050 - - ATPase complex/ soluble

Soluble protein

After in silico analyses, PtATPase1 with four potential TMDs and PtATPase2 without TMD were

selected for in vivo-localization analyses. The fluorescence pattern of PtATPase1-eGFP showed a dot-

like structure (Fig. 3-19a). In some clones several dot-like fluorescence could also be observed (Fig.

3-19b). By carbonate extraction it was shown that ATPase1 is an integral membrane protein (Fig. 3-

19d). It was suggested that ATPase1 might be localized on the small vesicles or vacuoles in P.

tricornutum. The second protein homologous to vacuolar H+-ATPase in A. thaliana is called ATPase2

in P. tricornutum. Based on the prediction the N-terminus of ATPase2 amino acid sequence does not

contain a hydrophobic signal peptide. When expressing the eGFP fusion construct it showed a

cytoplasmic localization, the fluorescence was distributed in the cell (Fig. 3-19c). By carbonate

extraction it was confirmed that ATPase2-eGFP is a soluble protein (Fig. 3-19e).

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Fig. 3-19: In vivo localization of V-ATPase proteins.

a/b: ATPase1-eGFP was expressed under the control of a nitrate inducible promoter. Slightly different fluorescence pattern

were observed, either one (a) or several (b) dot-like structures. ATPase2-eGFP showed a cytoplasmic localization. The

fluorescence was distributed in the cell. d/e: Carbonate extractions of ATPase1-eGFP and ATPase2-eGFP proteins,

respectively. The thylakoid membrane protein PsbD (25 kDa) and the stromal protein RbcL (55 kDa) were used as makers

for the fraction of soluble and integral membrane proteins. The expected molecular weight of ATPase1-eGFP and ATPase2-

eGFP are 45 kDa and 41 kDa, respectively. For a detailed description see text. TL: transmitted light; PAF (red): plastid

autofluorescence; eGFP (green): enhanced green fluorescent protein.

Discussion

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4 Discussion

4.1 Genetic compartmentalization of peridinin-containing dinoflagellates

Peridinin-containing dinoflagellates are important members of phytoplankton and, as stated in the

introduction, arose from secondary endosymbiosis of a red alga by a so far undefined host cell

(Mcfadden 2001). In contrast to other groups, their complex plastid is surrounded by only three

membranes (Dodge and Lee 2000). Apart from that peridinin-containing dinoflagellates stand out by

additional unusual features with regard to their genome organization (Zhang, Cavalier-Smith et al.

2002). Besides their extraordinary large nuclear genome which is organized in permanently

condensed, para-crystalline chromosomes (Bodansky, Mintz et al. 1979, Gautier, Michel-Salamin et

al. 1986, Bouligand and Norris 2001, Chow, Yan et al. 2010) they possess so called minicircles. Within

this thesis a new method transposon-insertion based approach for minicircle isolation from one

representative of the peridinin-containing dinoflagellates was used and finally isolated individual

minicircles were characterized.

4.1.1 Minicircles of the peridinin-containing dinoflagellate Amphidinium carterae CCAM0512

Since the discovery of minicircles of peridinin-containing dinoflagellats in 1999 (Zhang, Green et al.

1999) several minicircles of diverse peridinin-containing dinoflagellates were isolated and described

such as in A. operculatum, A. carterae CS21, A. carterae CCAP1102/6, Ceratium horridum, Adenoides

eludens, Heterocapsa species as well as Symbiodinium species (Zhang, Green et al. 1999, Barbrook

and Howe 2000, Barbrook, Symington et al. 2001, Hiller 2001, Zhang, Cavalier-Smith et al. 2002,

Moore 2003, Laatsch, Zauner et al. 2004, Nisbet, Koumandou et al. 2004, Nelson and Green 2005,

Barbrook, Santucci et al. 2006). Coding for genes normally found in conventional plastid genomes

were located on these small plasmid-like minicircles. Previous studies have already shown that the

minicircle contains one to three coding genes (coding region), the remainder of the minicircle was

called non-coding region (Barbrook and Howe 2000). It was also shown that several minicircles

without coding region do not have potential known function (also called empty minicircles) (Hiller

2001). Within this thesis a transposon-based method was used to isolate minicircles. So far,

transposons were predominantly used in bacteria, plants and animals for mutagenesis or some

other molecular biology studies (Kim, Vanguri et al. 1998, Balciunas and Ekker 2005, Carlson,

Frandsen et al. 2005, Schnable, Ware et al. 2009, Hackett, Largaespada et al. 2010). The transposon-

based approach is in so far different, that it is not based on known sequences and thereby has the

capacity to isolate minicircles with deviant or unknown sequence. The structural nature of plasmid-

Discussion

51

like minicircles provides the basis for transposon insertion and subsequent usage of bacterial

proliferation.

Within this thesis, by using the transposon-insertion based approach hundreds of thousands of

potential positive colonies grew on plastes. Based on the plasmid preparation and sequencing it was

found that 89 out of 107 (83.2%) sequencing samples are positive minicircles, 18 out of 89 (20.2%)

minicircles are gene-containing minicircles, 71 out of 89 (79.8%) minicircles are empty minicircles

(Table 3-1). The number of empty minicircles is about four times the number of gene-containing

minicircles. A possible explanation for this could be that the empty minicircles are present at higher

copy number than the gene-containing minicircles in A. carterae CCAM0512 cells. But this can not

rule out the possibility that the different division ratio of the E. coli cells harboring positive

minicircles in liquid LB medium without antibiotic (see materials and methods 5.2.4.12). 83.2%

sequencing sameples are positive minicircles, it was indicated that the transposon-insertion based

approach is a considerable efficient method. The isolated individual minicircles could work as a

vector and modified by different reporter gene (e.g. eGFP gene) for further research.

In the following points the implications deduced from the analysis of these different minicircles are

discussed.

4.1.1.1 Minicircles with coding genes

Of the isolated minicircles 18 out of 89 minicircles are gene-containing minicircles. Within these 18

minicircles it was found that 12 minicircles belong to different minicircle molecules. The 12

minicircles include three different psbA-containing minicircle molecules, two different petB/atpA-

containing minicircle molecules, two different 23S rRNA-containing minicircle molecules and one

psaB-, psbC-, psbD/E/I-, petD- and atpB-containing minicircle molecule (Table 3-2). These different

minicircle molecules exhibit open reading frames of considerable length which encode for known

proteins PsbA, PsaB, PsbC, PsbD, PsbE, PsbI, PetB, PetD, AtpA and 23S rRNA(Table 3-2). The lengths

of these individual minicircle molecules (2333 bps – 2664 bps) are similar to the sizes of minicircles

reported before such as A. carterae CS21 (2327 bps – 2713 bps), A. operculatum (2311 bps – 2713

bps) and H. triquetra (2151 bps – 3121 bps) (Zhang, Green et al. 1999, Zhang, Cavalier-Smith et al.

2002, Barbrook, Santucci et al. 2006). Compared to the minicircles in these species the length of

minicircles in Ceratium horridum are much larger 5200 bps - 6700 bps (Laatsch, Zauner et al. 2004).

The average length of eight different groups of gene-containing minicircles is 2474 bps. The variable

lengths of minicircle might result from the different evolutions and rearrangements of minicircles in

dinoflagellates. Most of the isolated minicircles are single-gene minicircles while the petB/atpA and

psbD/E/I gene pairs are encoded on one single minicircle of 2596 bps and 2354 bps, respectively.

Discussion

52

This is in line with the already reported two- or three- genes minicircles in other species (Barbrook,

Santucci et al. 2006). Previous studies showed that the petB/atpA genes, psbD/E/I genes on a single

minicircle are not normally adjacent to each other in reported conventional plastid genomes in other

plants and algae (Ohta, Matsuzaki et al. 2003, Puerta, Bachvaroff et al. 2005). The observed synteny

suggests that multiple genes on a single minicircle were generated by tandem rearrangement of two

or three single-gene minicircles, rather than by fragmentation of a multi-gene molecule (Howe,

Nisbet et al. 2008).

From the point of the gene transcription, the same as the transcriptions of genes that have been

studied previously (Zhang, Green et al. 1999, Barbrook and Howe 2000, Barbrook, Symington et al.

2001, Takishita, Ishida et al. 2004), it was shown here that the psbA gene might be also transcribed

in A. carterae CCAM0512. As for multiple genes minicircles previous studies suggested that these

genes (petB/atpA and psbD/E/I genes) on a single minicircle are transcribed separately as the sizes of

transcripts observed were the same as the single genes (Howe, Nisbet et al. 2008, Nisbet, Hiller et al.

2008), latter a polycistronic transcript was suggested which is produced first and subsequently

cleaved rapidly into two or three separate transcripts (Dang and Green 2010, Barbrook, Dorrell et al.

2012). It was demonstrated that the the minicircles were transcribed via a rolling circle model in the

dinoflagellates Hetercapsa triquetra and A. carterae CCAP1102/6 (Dang and Green 2010, Barbrook,

Dorrell et al. 2012). It was shown that the transcripts contain several ORFs which were not known

previously to be expressed (Barbrook, Dorrell et al. 2012). Simultaneously, Dang and Green showed

that the transcripts are even larger than the minicircle itself (Dang and Green 2010).

There is no doubt that the plastid genomes of dinoflagellates are the smallest compared to other

conventional plastid genomes. Previous studies suggested that this shrunken plastid genome might

stem from the transfer of the normally plastid-located genes to the host genome and dramatically

reducing and deleting of the chloroplast genome during the course of plastid acquisition (Bachvaroff,

Concepcion et al. 2004, Green 2004, Hackett, Yoon et al. 2004, Tanikawa, Akimoto et al. 2004,

Patron, Waller et al. 2005). Compared with all about 15 reported protein-coding genes on minicircles

psaA, psbB and small subunit ribosomal ribonucleic acid (SSU-rRNA) minicircles are still missing in

this work. Compared with 50 – 200 genes in other typical plastid genomes not only the tRNA but

genes encoding ribosomal proteins and several small proteins for photosynthesis and the ATPases

are still absence on known minicircles. A possible explanation for this could be that these genes have

already transferred into the nucleus. It’s also possible that these genes are not as abundant as the

other genes in A. carterae cells, thus it is hard to be isolated here. In initial studies putative f-Met

tRNA gene was identified in A. carterae CS21 and A. carterae CCAP1102/6 and putative proline and

tryptophan tRNA genes were found in Heterocapsa species (Barbrook, Santucci et al. 2006).

Discussion

53

Whereas tRNA gene was not found so far in A. carterae CCAM0512. It was shown that 15 retained

genes on minicircles encode subunits of the main complexes that involve in the light reactions of

photosynthesis such as photosystem I (PSI), PSII, the ATP synthase and the cytb6/f complex, as well

as rRNAs proteins (23S rRNA and 16S rRNA) and tRNA (Hiller 2001, Koumandou and Howe 2007,

Howe, Nisbet et al. 2008). All genes encoded on minicircles in A. carterae CCAM0512 use the

conventional genetic code with the same start codon (ATG) and three different stop codons (TAG,

TGA or TAA). In A. operculatum the psaA and psbB genes on minicircles do not have standard start

codon in the expected positions, while utilize GTA as an initiation codon (Barbrook and Howe 2000).

In terms of Heterocapsa triquetra ATA, TTG and ATG were used for the initial coding (Zhang, Green

et al. 1999). GTG becomes a potential start codon in petB gene containing minicircle in A. carterae

CS-21 (Hiller 2001). The variety of genetic codons might provide an important hint for evolutionary

relationship of minicircles.

RNA editing is a post-transcriptional process that can insert, delete and substitute nucleotides in

mRNA prior to translation to proteins. RNA editing can be widely observed on transcripts from

nuclear or organellar genomes (including mitochondrial and plastid) in different species (Miyata and

Sugita 2004, Wang and Morse 2006, Grosche, Funk et al. 2012, Takenaka, Zehrmann et al. 2013,

Mungpakdee, Shinzato et al. 2014). However, RNA editing is very rare to be observed in plastid-

encoded RNAs in algae. Only three editing events were found so far in the minicircle-encoded plastid

genes in Ceratium horridum, Lingulodinium polyedrum and Heterocapsa triquetra (Zauner, Greilinger

et al. 2004, Wang and Morse 2006, Dang and Green 2009). In contrast to previously reported

organellar RNA editing in peridinin-containing dinoflagellates, RNA editing was not observed on

transcriptions of minicircles of A. carterae CCAM0512 based on the analysis of nine coding

minicircles genes (eight genes were analysed by Grosche C.(Grosche 2012)).

Previous studies have already shown that the minicircles of dinoflagellates contain a highly

conserved core region (Zhang, Green et al. 1999, Barbrook, Symington et al. 2001, Zhang, Cavalier-

Smith et al. 2002). The core regions are species-specific and are unstable in length (Koumandou,

Nisbet et al. 2004). The highly diverse core regions of the four A. carterae strains indicated that the

core regions are only identical within the strain but considerable variation between different strains

(Fig. 3-3/7). The function of the core region remains uncertain. Based on the prediction the core

region has a putative promoter sequence in A. carterae CCAM0512 (Fig. 3-3). Previous studies

suggested that the initiation of replication usually possess multiple direct and inverted repeats in

plastids (Sears, Stoike et al. 1996, Kunnimalaiyaan, Shi et al. 1997). It was speculated that the core

region is responsible for the maintenance of the copy number, the initiation of replication and/or

the transcription of minicircles (Zhang, Green et al. 1999, Barbrook and Howe 2000, Barbrook,

Discussion

54

Symington et al. 2001, Hiller 2001, Zhang, Cavalier-Smith et al. 2002). It was also inferred to have a

function on the membrane attachment (Howe, Nisbet et al. 2008). As mentioned above, no obvious

direct or inverted repeat was observed in A. carterae CCAM0512. Therefore it is hard to speculate

the function of the core region.

4.1.1.2 Empty minicircles

In addition to the minicircles with obvious coding region, seventy-one empty minicircles were also

isolated (Table 3-3). Of the total 71 empty minicircles, 26 (36.6%) belong to the different empty

minicircle molecules. The 26 different empty minicircles were divided into six distinct groups

according to the conserved sequence. For example, the group of J8 empty minicircle contains three

different minicircle molecules in 17 minicircles. These empty minicircles have recognizable core

regions and include short open reading frames but potential products are without any known

homology. The average length of empty minicircle is 1986 bps, which is much smaller than gene-

containing minicircles (2474 bps). A direct reason is that the empty minicircles do not contain the

coding genes. The big difference of the lengths could also give a valuable hint for the regular

rearrangement of these minicircles during the evolution. Previous analysis showed that the

transcription of empty minicircles occurred in A. carterae CCAP1102/6 and A. carterae CS21 and this

transcription cover the large part of the minicircle including the core region (Nisbet, Hiller et al.

2008). As the transcriptions of three largest open reading frames in empty minicircles were observed

in A. carterae CCAM0512 (Fig. 3-4), the open reading frames of empty minicircles could be

transcribed and might be functional. Based on the comparison of the open reading frames (> 150

bps) it was found that empty minicircle J9 and atpB-containing minicircle have a completely identical

open reading frame and a partially identical open reading frame (Table 3-4). This could be explained

by that the empty minicircles were generated by the rearrangement between the gene-containing

minicircles. At the same time, it was shown that empty minicircles J13, J22 and J36 have an identical

open reading frame, empty minicircles J22 and J24 have an identical open reading frame. A possible

explanation for these identical open reading frames could be that the minicircles were generated by

tandem rearrangement of several different fragments during the evolutionary process, and these

fragments might be abundant in the cells. It could be speculated that these identical open reading

frames could be translated to functional proteins, but these proteins remain unknown so far.

A summary of GC content is shown in Fig. 3-5. Except the GC content of psbA-containing minicircle is

45%, which is the same as or lower than the GC content of the overall empty minicircles, all the

other gene-containing minicircles present to be more GC-rich than the empty minicircles. This

discrepancy might be important for the stability and expression of the coding regions.

Discussion

55

These empty minicircles may be unique to dinoflagellates. There remains many questions about the

empty minicircles: ‘how did they evolve? Where do they come from?’ and ‘What is the function of

the ORFs on empty minicircles? Do they translate?’ Nisbet et al. explained that the small empty

minicircles might generate from a homologous recombination and internal deletion event between

the minicircles with coding region (Nisbet, Koumandou et al. 2004). It was also speculated that

empty minicircles may come from ‘parasitic’ DNA elements (Howe, Nisbet et al. 2008). As the empty

minicircles do not have any identifiable coding characteristics, the functional analysis of these empty

minicircles remains hard.

4.1.2 The evolutional relationship of minicircles

So far, fourteen gene-containing minicircles were isolated from four A. carterae strains (Tab. 3-5), A.

carterae CCAM0512 (in this work), A. carterae CS21 (Hiller 2001, Barbrook, Santucci et al. 2006), A.

carterae CCAP1102/6 (Barbrook and Howe 2000, Nisbet, Koumandou et al. 2004) as well as A.

carterae CCMP1314 (Zhang, Cavalier-Smith et al. 2002).

Our present results revealed that the core regions are highly identical in A. carterae CCAM0512 (Fig.

3-3), but the core regions of four A. carterae strains are apparently unrelated and cannot be

mutually aligned (Fig. 3-7). An alignment of the non-coding regions of the four psbA minicircles

showed that upstream and downstream the psbA gene are conserved. The conserved regions might

be useful for the stability of the gene expression (Fig. 3-8). Together with this, a conserved sequence

of 50 bps upstream of the psbA gene and downstream of the core regions might have a function on

the transcription initial based on the prediction. It was shown that the core regions of these four A.

carterae strains are located in the variable region. On the contrary, the coding regions (e.g. psbA

minicircles) of these four A. carterae strains showed a considerable high identity (more than 97%)

based on the molecular phylogenetic analysis (Fig. 3-6). From the phylogenetic analyses of LSU rDNA

and SSU rDNA sequences, these four A. carterae strains were found to be a sister relationship in a

clade containing A. carterae with high statistical support (Fig. 3-9 and Fig. 3-10).

Heretofore, several empty minicircles were identified including 10 empty minicircles in A. carterae

CS21 and 5 empty minicircles in A. carterae CCAP1102/6 (Barbrook, Santucci et al. 2006). Within this

thesis, six distinct groups of empty minicircles were identified in 26 different empty minicircle

molecules in A. carterae CCAM0512. Among all small open reading frames 27 open reading frames

(the length > 150 bps) were compared with that in A. carterae CS21 and A. carterae CCAP1102/6

(Table 3-6). It was found that 7 out of 27 pairs of open reading frames have a high query cover, low

e-value and high identity. Among these three pairs of open reading frames have a 100% query cover,

very low e-value and considerable high identity (83%, 98% and 96%). It was speculated that these

Discussion

56

open reading frames might have a specific function in dinoflagellates, but it has to be comfirmed in

the future. The presence of conserved open reading frames among these different A. carterae

strains suggested that these open reading frames are evolutionary conserved and are related to

each other.

Taken all these analyses together, it was shown that nuclear encoded LSU and SSU and minicircle

encoded psbA are highly identical in these four A. carterae strains, while the core regions are

completely different. A hypothesis was put forward to suggest that the core regions of minicircles

evolved at a very fast speed. As the core regions are highly diverse within the different strains, this

could be used to efficiently distinguish different strains in dinoflagellates such as the toxic and non-

toxic strains.

Discussion

57

4.2 “The endomembrane system (ES) in Phaeodactylum tricornutum”

As already mentioned the endomembrane system is made up of the different organellar membranes

including the nuclear envelope, ER, Golgi apparatus, lysosomes or vacuoles, vesicles, endosomes and

the plasma membrane. In eukaryotic cells, proteins are encoded in the nuclear genome and

synthesized in the cytoplasm, some of them must be transported to the different subcellular

compartments such as the plasma membrane, lysosomes/ vacuoles or the extracellular. Only if the

proteins are targeted to their appropriate final destinations, they can perform required function.

However, the mechanisms for trafficking of proteins during the endomembrane system in vivo in the

diatom P. tricornutum remain poorly understood. The known marker proteins in P. tricornutum are

not enough. Consequently, the aim of this project was to identify different marker proteins localized

in the different subcellular compartments and therefore provide an essential condition for further

detailed researches about the protein trafficking on endomembrane system.

4.2.1 Identification of tonoplast intrinsic proteins (Tips)

Water and other small molecules across the membrane is largely controlled by membrane channels

called tonoplast intrinsic proteins (Tips) (Höfte, Hubbard et al. 1992). Tips together with another four

groups the plasma membrane intrinc proteins (PIPs), nodulin 26-like intrinsic proteins (NIPs) and

small basic intrinsic proteins (SIPs) belong to the aquaporins family. The aquaporins are channel

proteins belong to the major intrinsic protein family (MIPs) (Johanson and Gustavsson 2002).

Subcellular localization of several Tip isoforms (including three gamma-Tip (Tip1), three delta-Tip

(Tip2), the seed specific alpha- and beta-Tip (Tip3:1 and Tip3:2), one epsilon-Tip (Tip4:1) and one

zeta-Tip (Tip5:1)) have already been identified or predicted in vivo in A. thaliana (Höfte, Hubbard et

al. 1992, Johanson, Karlsson et al. 2001). Previous study has already shown that these channel

proteins are distributed to specific developmental stages and tissue types in plant cells (Rivera-

Serrano, Rodriguez-Welsh et al. 2012). It was identified that Tip1:1 (gamma-Tip) and Tip2:1 (delta-

Tip) proteins are targeted to the tonoplast of the central vacuole in transgenic A. thaliana mature

roots, root tips and leaves (Hunter, Craddock et al. 2007). While in embryos of seeds Tip1:1 and

Discussion

58

Tip2:1 are transported to the protein storage vacuoles (Karlsson, Johansson et al. 2000, Saito, Ueda

et al. 2002, Gillespie, Rogers et al. 2005, Hunter, Craddock et al. 2007, Schüssler, Alexandersson et al.

2008, Gattolin, Sorieul et al. 2009). Subcellular localization of eGFP-fused Tip protein indicated that

AtTip4:1 and AtTip1:2 were localized mainly on the tonoplast membrane and other uncharacteristic

endomembranes (Liu, Ludewig et al. 2003).

Within this thesis an initial localisation for five Tip proteins homologous to Tips of A. thaliana is

present first in P. tricornutum. The use of eGFP and mRFP fusions to these Tips cDNA sequences

allowed us to investigate the subcellular localization in vivo. It was shown that Tip1-eGFP was

obviously targeted to the plasma membrane in P. tricornutum, and additional dot-like fluorescence

was also observed in some clones as shown in Fig. 3-11. However, the fluorescence pattern of the

Tip1-eGFP was not completely consistent with the fluorescence of plasma membrane protein-PDZ2,

which was solely and equally distributed to the plasma membrane (Stork 2013). In order to explain

the localization of these dot-like structures co-expression of Tip1-eGFP with FM4-64 was performed.

FM4-64 is specific used for staining the plasma membrane and to follow endomembrane system-

dependent internalization processes (Rigal, Doyle et al. 2015). The overlapping of these dots with

dots stained by the dye FM4-64 indicated that the dot-like structures belong to the endomembrane

system. Based on these results, Tip1-eGFP fusion was speculated to localize on the plasma

membrane and endosomal vesicles. This is important and the first time to show the recycling of

plasma membrane proteins in diatoms. It is still unknow whether the endocytosis happens in

diatoms or not. The identification of the recycling plasma membrane in diatoms is very useful for the

endocytosis research. The subcellular localization of Tip2 showed that Tip2-full length-eGFP fusion

protein was typically targeted to the vacuolar-like membrane, as shown in Fig. 3-12. As the model of

Tip2 is not completely supported by expressed sequence tags (ESTs), the Tip2 gene was amplified

from cDNA. The alignment of Tip2 nucleotide sequences see supplements 7.3. Tip2 is the first

identified vacuolar-like marker protein in diatoms in P. tricornutum. Thus, it is very important for

investigating the mechanisms of vacuolar protein transport. At the same time, according to the

predictions the N-terminus of Tip2 protein sequence is lack of signal peptide, and Tip2 contains six

conserved transmembrane domains, three extra loops and two-hydrophobic intracellular loops.

Therefore, it will be very meaningful and interesting to mutate several potential targeting signal for

studing the secretory pathway of Tip2, such as the mutations of the N-terminal amino acid, C-

terminal amino acid and the amino aicd on the their loops.

Endosymbiotic events gave rise to the formation of different groups of organisms with the so-called

complex or secondary plastids. The complex or secondary plastid is surrounded by four membrane

structures in P. tricornutum. The outermost membrane of the complex plastid called chloroplast ER

Discussion

59

(cER membrane) membrane, it is continuous with the outer membrane of the nuclear envelope and

was thought to connect with the host ER (Gibbs 1979, Cavalier-Smith and Chao 2003). Subcellular

localization of eGFP-fused Tip3 and Tip5 indicated that they localized on the outermost membrane

of the complex plastid, nuclear envelope and host ER (Fig. 3-13). It was observed that the immuno

gold particles are predominantly present in the outermost membrane of the plastid (cER membrane)

and partially present in the nuclear envelope and host ER by applying the electron microscopy.

Based on the absence of N-terminal targeting signal sequence (BTS) in both Tip proteins, it was

further indicated that Tip3 and Tip5 locate on the cER membrane. In comparison with an important

component of the host ERAD machinery (cER membrane, nuclear envelope and host ER marker-

hDer1) (Hempel, Bullmann et al. 2009), it was observed that to some extent the fluorescence

pattern of three proteins are similar. Co-expression of Tip3-mRFP with hDer1-eGFP showed the

overlapping of the fluorescence but the distribution of the fluorescence is different. Taken all these

results together, Tip3- and Tip5-eGFP fusions were transported to the outermost membrane of the

complex plastid (cER membrane), nuclear envelope and host ER in P. tricornutum (Fig. 3-13/14). All

these makers are important for investigating protein transport across these ER membranes. These

abundant ER membrane markers provide a valuable insight for distinguishing different structures of

ER membranes and better investigating the function of these ER structures.

Subcellular localization of eGFP-fused Tip4 indicated that Tip4 was inserted into the second

outermost membrane (PPM). Several facts support this speculation. Firstly, the fluorescence pattern

is typical PPM localization, which is similar to the fluorescence distribution of the identified PPM

marker PtE3P (Hempel, Felsner et al. 2010). Secondly, Tip4-eGFP is a membrane protein by the

carbonate extraction, as shown in Fig. 3-15. Thirdly, the self-assembly GFP assay indicated that the

C-terminus of Tip4 is localized to the PPC. Last but not the least, Tip4 is the only one identified

aquaporin protein of these five Tip homologous proteins with a predicted signal peptide and an N-

terminal extension, which could lead the protein transport into the PPC or the PPM. Previous study

showed that only at the +1 position of the potential signal peptidase cleavage site is an aromatic

amino acid, the plastid protein is allowed to import (Gruber, Vugrinec et al. 2007). However, at the

+1 position of the Tip4 potential signal peptidase cleavage site is not an aromatic amino acid. Taken

all these evidences together, Tip4 should be targeted to the PPM of the complex plastid.

Discussion

60

Fig. 4-1: Schematic overview of identified marker proteins in diatom P. tricornutum.

Proteins which have been identified within this thesis were shown in this figure. The markers of different subcellular

compartments include five Tips, Tip1 (plasma membrane (PM) and endosomal compartments), Tip2 (vacuolar-like

structure), Tip3 and Tip5 (nuclear envelope, cER and host ER (hER) membranes) and Tip4 (second outermost membrane of

the complex plastid, PPM), two subunits of the putative retromer Vps26 and Vps29 (trans Golgi network), three Golgi

apparatus markers GnTI (cis Golgi network), XylT (medial Golgi network) and FucT (trans Golgi network), and two V-

ATPases ATPase1 (probably in a second type of vacuole) and ATPase2 (cytosol). Nu: nucleus, Mit: Mitochondria.

All in all, the subcellular localizations of five Tips (Tip1-5) were addressed in P. tricornutum (Fig. 4-1).

Contrary to the Tips in higher plants five Tips was not only targeted to the tonoplast in P.

tricornutum. Tip1 is targeted to the plasma membrane and endosomal vesicles, Tip2 is located on

the vacuolar-like structure, Tip3 and Tip5 are enriched on the cER membrane, nuclear envelope and

host ER, Tip4 is a PPM protein. These results indicated that the subcellular localizations of the

protein candidates are not always matched with the predictions, the localizations could be

completely different in different organisms. A possible explanation for this could be that not all

these five Tips are really homologous to tonoplast intrinsic proteins in higher plants, such as Tip1

might be homologous to one of the plasma membrane intrinsic proteins (PIPs) or the nodulin 26-like

intrinsic proteins (NIPs) which localized in the plasma membrane and ER in higher plants, Tip3 and

Tip5 might be homologous to the small basic intrinsic proteins which localized in the ER in higher

plants (Johansson, Karlsson et al. 2000, Quigley, Rosenberg et al. 2002, Pandey, Sharma et al. 2013).

Another possible explanation for this could be that the expression of these Tips relies on the specific

developmental stages and tissue types in higher plants (Rivera-Serrano, Rodriguez-Welsh et al. 2012).

The proteins expressed in different conditions might effect their localizations. Thus, it can not be

ruled out the reason from the different organisms. About four hundred aquaporins were identified

Discussion

61

in recent study in ten different plants containing monocots and dicots (Regon, Panda et al. 2014).

Among them, Citrus sinensis (57), Fagaria vesca (42), Sorghum bicolor (40) and Zea mays (43) have

higher aquaporin genes than the others. 38 aquaporin genes and 11 Tips were identified in A.

thaliana (Regon, Panda et al. 2014). Regarding to the diatom P. tricornutum, 42 major intrinsic

proteins were found in the database. So far, only one Tip protein (Tip2) homologous to Tip of A.

thaliana is a tonoplast intrinsic protein in P. tricornutum.

Previous studies have already shown that the localizations and amount of Tips in higher plants could

be changed by stressed conditions such as the salt exposure and dark adaptation (Boursiac, Chen et

al. 2005, Uenishi, Nakabayashi et al. 2014). Thus, the identified five Tips in P. tricornutum could also

be used to study the expression and localization in different environmental stress conditions.

Compared with most aquaporins in higher plants, it was shown that these five Tips also have either

one or two NPA motifs in their sequences and six transmembrane domains (Table 3-7). To some

extent, this suggested that all the Tips are closely related and conserved during the process of

evolution. The mutation of these NPA motifs will be a method to detect its function. The function of

these five Tips remains unknown in P. tricornutum. It is still unknown whether they could form an

aquaporin pore to selectively mediate the transport of water, gases and small neutral solutes.

Therefore, the identification of the localizations of these candidate proteins will be very important

for the studying of these proteins’ function.

4.2.2 Identification of Golgi-marker proteins

In most eukaryotes, the Golgi apparatus is divided into the cis-Golgi apparatus, the medial Golgi

apparatus and the trans-Golgi apparatus. As already mentioned in the introduction the N-

glycosylation pathway mainly occurs in the ER and the Golgi apparatus (Rayon, Lerouge et al. 1998,

Mathieu-Rivet, Kiefer-Meyer et al. 2014). This process is catalyzed and modified by a large number

of important and highly conserved membrane-bound glycosylhydrolases and glycosyltransferases

such as GnT1, XylT and FucT. The N-glycosylation pathway is mainly-studied in yeast and higher

eukaryotes, but the data regarding the glycosyltransferases localization in this pathway remain

unknown in diatoms. Three proteins homologous to AtGnT1, AtXylT and AtFucT were fused to eGFP

and expressed in vivo P. tricornutum. It was shown that the sequence of PtXylT shares 24% identity

with the AtXylT (Baïet, Burel et al. 2011). As expected these three eGFP fusion proteins showed

similar dot-like or long strip-like fluorescence pattern (Fig. 3-16). This could be explained by the Golgi

localization of the three candidate proteins via the known localization in plant. In A. thaliana GnT1 is

known to be localized in the cis-Golgi apparatus (Kajiura, Okamoto et al. 2012). AtXylT was targeted

to the medial cisternae of Golgi (Pagny, Bouissonnie et al. 2003, Kajiura, Okamoto et al. 2012). FucT

Discussion

62

is localized to the trans-Golgi or TGN in A. thaliana (Fitchette‐Lainé, Gomord et al. 1994). The N-

glycosylation in mammalian is different. The GnT1 is substituted by an alfa-1, 3-fucose. The XylT and

FucT transferases locate on the trans-Golgi apparatus (Kim, Jeon et al. 2014).

In some cases banana-like fluorescence pattern of XylT-eGFP was observed, as shown in Fig. 3-16b.

The fluorescence pattern matches with the shape of the Golgi apparatus. Based on the co-

expressions of XylT-mRFP with GnT1-eGFP and XylT-mRFP with FucT-eGFP, it was observed that the

fluorescence overlapped only partially. Taken together, it was indicated that the localizations of

these three Golgi marker proteins are slightly different. Previous study indicated that AtXylT acts at

multiple stages of the plant N-glycosylation pathway, especially in the medial-Golgi (Kajiura,

Okamoto et al. 2012). This could be a reason to explain why the XylT could partially overlap with the

cis-Golgi marker GnT1 and the trans-Golgi marker FucT in P. tricornutum. Therefore, it was

speculated that GnT1 might be localized on the cis Golgi apparatus, XylT is majorly targeted to the

medial Golgi apparatus, while FucT is more possible enriched in the trans Golgi apparatus (Fig. 4-1).

However, it is still unknown whether these three potential Golgi markers could also work in the

course of N-glycosylation in P. tricornutum or not. In any case, the identification of the three Golgi

marker proteins is important for the studying on their function and the pathway of N-glycosylation

in P. tricornutm. N-glycosylation is an important and ubiquitous modification in the synthesis of new

proteins in eukaryotes. This modification occurs in the course of protein secretion. It is crucial for the

right folding, structural formation and assembly of the secreted proteins and the precisely targeting

of glycoproteins to outside the cell or the membranes. Thus, the mutation of the glycosylation sites

will be a new insight to investigate the protein transport in diatoms. As β1, 2-xylosylated N-glycans

might have a function on inducing immune-reponses as pollen allergens which indicates that β1, 2-

xylosylated N-glycans are equivalently interest in the algal-produced biopharmaceuticals.

4.2.3 Identification of retromer complex proteins

In addition to the already described coat proteins the retromer complex is mainly responsible for the

retrograde transport of protein sorting receptors (Seaman 2005, Bonifacino and Hurley 2008,

Schellmann and Pimpl 2009, Reyes, Buono et al. 2011). The retromer complex contains two

subcomplexes, a large subcomplex is formed by three core subunits (Vps26, Vps29 and Vps35) for

cargo recognition and a small subcomplex is formed by membrane deforming sorting nexin proteins

(SNXs) in yeast, plants and mammal cells (Bonifacino and Hurley 2008, Otegui and Spitzer 2008,

Cullen and Korswagen 2012). The structure of retromer has been well-studied in yeast, plants and

mammal cells, but the localization of the core subunits and sorting nexins of retromer is still debated.

Discussion

63

Within this thesis the subcellular localization of two homologous proteins to large retromer subunits

of A. thaliana Vps26 and Vps29 were investigated in vivo in P. tricornutum. As expected the

fluorescence patterns of the Vps26- and Vps29-eGFP fusion protein are similar, a dot or small

diamond-like structure (Fig. 3-17). The co-localization of the Vps26-eGFP and Vps29-mRFP indicated

that these two homologous proteins located in the same structure and might also belong to the

retromer complex in P. tricornutum. In the present investigation, where the co-localization of the

Vps29-mRFP with the TGN marker FucT but only partially overlap of the Vps29-mRFP with the medial

Golgi marker XylT was observed. In agreement with recent studies in Arabidopsis and tobacco roots

(Niemes, Langhans et al. 2010, Stierhof, Viotti et al. 2013) Vps26 and Vps29 subunits are localized to

the TGN in P. tricornutum (Fig. 4-1). This provides an important prerequisite for the investigation of

the function of retromer complex and the relationship between the retromer and three Golgi maker

proteins. The receptor-dependent protein transport between different intracellular compartments is

essential for many physiological activity in plant. The continuous recycling of receptors via retromer

evade the degradation and can be used for the next rounds of protein transport. Previous studies

have already idendified that trans Golgi network is the starting point for receptor-ligand interaction

and package the receptor-ligand complexes into the clathrin-coated vesicles, subsequently, the

cargo will be delivered into the next comparments such as the prevacuolar compartments/ late

endosomal compartments (Tse, Mo et al. 2004, Detter, Hong-Hermesdorf et al. 2006, Robinson,

Jiang et al. 2008). To some extent, it is contradicts to the current localization of the retromer.

The localization of retromer in higher plant cells is debated. The localization of retromer subunits

seems not to be restricted to the TGN. Previous studies showed that the retromer in mammals

localized to the tubular extensions of early and recycling endosomes (Carlton, Bujny et al. 2004,

Bonifacino and Hurley 2008). Published data showed that the sorting nexins and the components of

the large retromer subunit proteins were localized to the pre-vacuolar compartment (PVC) in A.

thaliana and tobacco roots (Geldner, Anders et al. 2003, Tse, Mo et al. 2004, Oliviusson, Heinzerling

et al. 2006, Jaillais, Santambrogio et al. 2007, Kleine-Vehn, Leitner et al. 2008, Yamazaki, Shimada et

al. 2008). It was also suggested that the SNX2b and SNX1 are localized to the TGN, PVC and an

endosomal compartment in plant (Phan, Kim et al. 2008, Robinson, Jiang et al. 2008). However, the

colocalization of SNX2a with TGN markers but not with Golgi or PVC marker indicated that the

sorting nexins are exclusively localized to the TGN rather than the pre-vacuolar compartment in

Arabidopsis and tobacco roots (Niemes, Langhans et al. 2010). Previous studies showed that the

retromer complex consists of sorting nexins subcomplex and the large Vps26/29/35 core subunits,

but later it was suggested that the two subcomplexes may not always bind together and might have

different functions in A. thaliana (Harbour, Breusegem et al. 2010, Pourcher, Santambrogio et al.

2010). This makes the localization of retromer subunits even more difficult.

Discussion

64

4.2.4 Identification of vacuolar type H+-ATPases

In order to identify more vacuolar marker protein, the vacuolar type H+-ATPases (VHAs) were

analyzed in silico. This membrane-bound multisubunit complex contain at least 26 genes encoding

subunits (Sze, Schumacher et al. 2002). VHAs are essential for mediating the pH of intracellular

compartments. Simultaneously, VHAs are important for protein transport, plant growth,

development and adaptation to the changing environmental conditions and maintaining metabolite

and ion balance in plant cells (Sze, Schumacher et al. 2002, Kluge, Lahr et al. 2003). Inhibition of the

vacuolar type H+-ATPase effect the secretion and results in the mistargeting of vacuolar proteins in

plant (Matsuoka, Higuchi et al. 1997).

The fluorescence pattern of PtATPase1-eGFP fusion protein showed one or in some clones three

dot-like structures in P. tricornutum (Fig. 3-19a/b). Based on the hypothesis VHA-c is targeted to the

ER, TGN and the vacuole-specific subsector in A. thaliana (Seidel, Schnitzer et al. 2008). PtATPase1

homologous to hydrophobic subunit C of A. thaliana is a membrane protein. Thus, it was speculated

that these dot-like fluorescence might be localized on the small vesicles or small vacuoles.

Another protein homologous to VHA-G2 subunit of A. thaliana is PtATPase2. PtATPase2-eGFP fusion

protein showed a cytosolic fluorescence (Fig. 3-19c). The best match protein in A. thaliana is

vacuolar ATPase protein 10 (TAIR: AT3g01390.2). It belongs to the subunit G of vacuolar type ATPase

cytosolic V1 sector. The identified cytosolic PtATPase2 is in agreement with the published data from

A. thaliana (Aviezer-Hagai, Nelson et al. 2000, Endler, Meyer et al. 2006). As already shown in the

introduction the vacuolar type H+-ATPase contains at least 26 gene encoding subunits, therefore, the

localization of the two subunits is only the starting point in P. tricornutum. The identification of the

subcellular localizations of these VHA subunits might be very useful for further studies on the

localization of the other VHA subunits and for researches on the function of VHAs in the diatom P.

tricornutum.

Materials and Methods

65

5 Materials and Methods

5.1 Materials

5.1.1 Instruments

PCR-Thermo-Cycler:

Mastercycler gradient Eppendorf, Hamburg Mastercycler personal Eppendorf, Hamburg

Centrifuges:

Centrifuge 5415 D Eppendorf, Hamburg Centrifuge 5417 R Eppendorf, Hamburg Centrifuge 5810 R Eppendorf, Hamburg Mikro 22 R Hettich Zentrifugen, Tuttlingen MiniSpin® Plus Eppendorf, Hamburg PicoFuge® Stratagene, La Jolla, USA L755 Ultracentrifuge Beckman Coulter

Biolistic transfection:

FrenchPress MiniZelle FA-003 G. Heinemann ULT Biolistic PDS-1000/He Particle Delivery System

Biorad, Munich

Rupture Discs 1350 psi Biorad, Munich Macrocarrier Biorad, Munich M 10 (Ø 0.7 μm) Tungsten-Particles Biorad, Munich Frenchpress SLM-AMINCO 4-3399 SLM-AMINCO Instruments

Confocal Laser Scanning Microscope:

CLSM Leica TCS SP2 Leica, Wetzlar

Incubation:

Incubator Heraeus Instruments, Hanau Climate Chamber MLR-350 SANYO Ewald Gmbh Thermocycler 60 Biomed, Oberschleißheim Thermomixer comfort Eppendorf, Hamburg Thermomixer compact Eppendorf, Hamburg


66

Other instruments:

Nanodrop ND-1000 photometer peqlab ABI Prism 377 Applied Biosystems

5.1.2 Membranes and filters

Nitrocellulose membrane Macherey-Nagel Whatman 3MM Schleicher & Schuell, Dassel FP 30/ 0.2 CA-S - 0.2 μm sterile filter Schleicher& Schuell Fuji-Medical-X-ray-Film, 30 x 40 cm Fuji Film X-ray film developer and replenisher Kodak

5.1.3 Antibodies

Primary antibodies Dilution Manufacturer α GFP 1:3000 Biomol α Rubisco 1:5000 Agrisera α psbD(D2 protein of PSII) 1:5000 Agrisera α psbO 1:1000 Agrisera Secondary antibodies Dilution Manufacturer αgoat (HRP coupled) 1:10000 Sigma-Aldrich, Munich αrabbit (HRP coupled) 1:10000 Sigma-Aldrich, Munich

5.1.4 Chemicals

Unless otherwise noted, all chemicals used in this work were obtained from Roth GmbH, Sigma

Aldrich or Merck and stored and used according to the manufacturer’s instructions.

5.1.5 Enzymes

DNAseI Thermo Scientific/Fermentas, St. Leon-Rot Phusion High Fidelity DNA-Polymerase (5 U/µl)

Thermo Scientific/Fermentas, St. Leon-Rot

Restriction endonucleases (10 U/µl) Invitrogen, Karlsruhe RNase A (70 U/μl) Thermo Scientific/Fermentas, St. Leon-Rot T4-DNA-Ligase (1 U/μl) Thermo Scientific/Fermentas, St. Leon-Rot Taq-DNA-Polymerase Biotools, Madrid


67

5.1.6 Software and bioinformatic applications

Sequencher 5.1 GeneCodes LCS Lite 2.5 Leica ClustalW and ClustalX Alignment Mega4.0 and Mega6.0 Phylogenetic analyses ImageJ Tony Collins, McMaster Biophotonics Facility

The following websites were commonly used:

SOSUI http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html

TMHMM Server v.2.0 http://www.cbs.dtu.dk/services/TMHMM-2.0/ ΔG prediction server v1.0 http://dgpred.cbr.su.se/index.php?p=fullscan TOPCONS http://topcons.cbr.su.se/ SignalP 4.1 Server http://www.cbs.dtu.dk/services/SignalP/ TargetP 1.1 Server http://www.cbs.dtu.dk/services/TargetP/ ChloroP Server http://www.cbs.dtu.dk/services/ChloroP/ Psortb http://www.psort.org/psortb/ Phaeodactylum digital gene expression

http://www.diatomics.biologie.ens.fr/EST/est.htm

P. tricornutum data base v2.0 http://genome.jgi-psf.org/pages/blast.jsf?db=Phatr2 Tm calculator http://www.thermoscientificbio.com/webtools/tmc/ Pubmed http://www.ncbi.nlm.nih.gov/entrez ClustlW2 http://www.ebi.ac.uk/Tools/clustalw2/index.html Clustal Omega http://www.ebi.ac.uk/Tools/msa/clustalo/ TransportDB http://www.membranetransport.org/ Arabidopsis thaliana http://www.arabidopsis.org

5.1.7 DNA and protein markers

For agarose gel electrophoresis, the GeneRulerTM 1 kb Plus DNA Ladder or Lambda

DNA/EcoRI+HindIII Marker from Thermo Scientific/MBI Fermentas were used as DNA length marker.

As for SDS-PAGE marker, the PageRulerTM Prestained Protein Ladder from Thermo

Scientific/Fermentas was used.

5.1.8 Oligonucleotide primers

All oligonucleotide primers used in this work were supplied from Sigma. All oligonucleotide primers

used for amplifying genes, colony PCR and sequencing in this work were listed in supplemental data

7.3. The working concentration of primers was 5 pmol/µl.

http://dgpred.cbr.su.se/index.php?p=fullscan

http://www.ncbi.nlm.nih.gov/entrez

http://www.arabidopsis.org/


68

5.1.9 Vectors

pJet1.2/blunt cloning vector Ampr,PT7, eco47IR MBI-Fermentas pPha-T1 (provided from Peter Kroth, University Konstanz, Germany)

Ampr, Zeor,PfcpA +TfcpA Zaslavskaia et al.2000

pPha-NR Ampr, Zeor, PNR+ TNR Acc. JN180663 pMOD™-3 <R6Kγori/MCS> Ampr, R6Kγori Cat.No.MOD1503 pPha-Dual 2xNR Ampr, Zeor, PNR+TNR(both MCS I

and II) Acc. JN180664

5.1.10 organisms

Escherichia coli TOP10 (F-, mcrA, Δ(mrrhsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, nupG, recA1,

araD139, Δ(ara-leu)7697, galE15, galK16, rpsL(StrR), endA1, λ) from Invitrogen.

Phaeodactylum tricornutum, Strain CCAP 1055/1

Amphidinium carterae strain CCAM0512

TransforMaxTMEC100DTM Pir-116 Electroncompetent E.coli

F– mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU

galK λ– rpsL nupG pir-116(DHFR). Maintains plasmids at ~250 copies per cell.

TransforMax EC100D pir+

F– mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU

galK λ– rpsL nupG pir+(DHFR). Maintains plasmids at ~15 copies per cell.

5.2 Methods

5.2.1 Culture of E.coli TOP10

E.coli TOP10 cells were obtained from Invitrogen, Karlsruhe. For long-termstorage, E.coli TOP10 cells

were stored at -800C in a LB/glycerin (1:1) mixture. Short-termcultivation was carried out overnight

at 37°C and constant shaking (200 rpm) insterile LB liquid medium or on 1.5% LB-agar plates with the

appropriate antibiotic (50 µg/ml ampicillin or 25 µg/ml kanamycin).

LB-medium: (pH=7.0) 1% (w/v) Bacto-Tryptone 0.5% (w/v) Yeast extract 1% (w/v) NaCl 50 µg/ml ampicillin or 25ug/ml kanamycin Add 1.5% (w/v) agar for LB solid medium.


69

5.2.2 Culture of Phaeodactylum tricornutum CCAP 1055/1

P. tricornutum cells were cultured under 24 h light condition (8000-11000 1x) at 22°C, constantly

shaken at 225 rpm in f/2- liquid medium or on f/2 agar plates. The cultures were inoculated each

two to three weeks for strain maintenance. For the selection of transformed clones, a concentration

of 75 μg/mL zeozinTM (InvivoGen) was added to the medium. For strain maintenance cultures were

transferred to new f/2 agar plates every four weeks.

f/2 medium (pH 7.0) Tropic marine sea salt 16.6 g Tris (2 M, pH 8.0) 1 mL NaH2PO4 · H2O (0.1 M) 360 μL NaNO3 (1 M)/ NH4Cl 890 μL/ 1 mL f/2 vitamin solution 1 mL Trace elements 1 mL ad 1 L dH2O

5.2.3 Culture of Amphidinium carterae CCAM0512

Amphidinium carterae (Hulburt) (strain CCAM0512) comes from the algae collection department of

Marburg University and were cultured in f/2-medium at 12 h/ 12 h light/ dark photoperiod, 20 °C.

For putative minicircles isolation, the cells were cultured to the middle of stationary phase.

f/2 medium (pH 7.0)

Tropic marine sea salt 30 g

Tris (1 M, pH 8.0) 5 ml

NaH2PO4 · H2O (0,1 M) 360 μl

NaNO3 (1 M) 900 μl

f/2 vitamin solution 1 mL

Trace elements 1 mL

ad 1 L dH2O

f/2 trace elements f/2 vitamin solution

FeCl3 11.65 mM Biotin 2 μM

Na2EDTA 11.71 mM Cyanocobalamine 0.37 μM

CuSO4 39 μM Thiamine-HCl 297 μM

ZnSO4 77 μM

CoCl2 42 μM

MnCl2 910 μM

Na2MoO4 26 μM


70

5.2.4 Nucleic acid analytics

5.2.4.1 Plasmid isolation from E.coli

Plasmid isolation from E.coli was carried out by alkaline extraction according to (Bimboim and Doly

1979). Previous to plasmid preparation, liquid cultures were inoculated from colonies grown on LB

agar plates and incubated overnight at 37°C. The next day, 1.5ml of the liquid culture was used and

centrifuged full speed for 1min. After discarding the supernatant by tilt or tap, the pellet was

resuspended in 200 μl of P1 buffer. 200 μl of buffer P2 were added and mixed with the suspension

by inverting the Eppendorf cup 5-10 times. Following an incubation for 5 min at RT, 200 µl of P3 and

20 µl chloroform were added and mixed with the lysate. After an incubation of 5 min on ice the

lysate was centrifuged for 5 min (4°C/ 20,000 g). The resulting supernatant was then transferred to a

new Eppendorf cup and 400 µl of isopropyl alcohol was added and mixed with the supernatant.

Following a centrifugation (20 min/ 4°C/ 20,000 g) the supernatant was discarded and the pellet

washed with 70 % ethanol (5 min/ 4°C/ 20,000 g). The supernatant was again removed and the dried

pellet was finally dissolved in 40 µl ddH2O.

Buffer P1 Buffer P2 Tris/HCl (pH 8.0) 50 mM NaOH 200 mM EDTA 10 mM SDS 1 % (w/v) RNase A 100 μg/mL Buffer P3

KAc (pH 5.5) 3 M

5.2.4.2 DNA and RNA isolation from P. tricornutum

The isolation of genomic DNA from P. tricornutum was carried out via the CTAB-method described

(Doyle and Doyle 1990). P. tricornutum cells were collected from 150 mL liquid culture by

centrifugation (3000 g/ 5 min/ RT). The supernatant was discarded and pellet was resuspended in

1600 μL of 2x CTAB containing buffer B. Cell lysis, then the suspensions was transferred into two

new Eppendorf cups (EP) and incubated at 70 °C for 30 min, after that cell debris was centrifuged

(20000 g/ 5 min/ RT). The upper phase was moved to a new EP and mixed with one volume of PCI

(DNA isolation, saturated with EDTA) gently, then centrifuged at 20000 g/ 10 min/ RT. Subsequently,

the upper phase was moved to a new EP again, mixed with 1/10 volume of NaAc (pH 4.5) and 2/3

volume of isopropyl alcohol gently and centrifuged (20000 g/ 20 min/ 4 °C). After that, the

supernatant was removed and the pellets was washed with 500 μL of 70 % ethanol and centrifuged

(20000 g/ 20 min/ 4 °C). At last, the supernatant was discarded, while the pellets were dried in a


71

desiccator. After drying, the pellet was dissolved in 15 μL of ddH2O, after which the DNA solutions

were transferred into one EP.

RNA isolation was carried out by collecting 150 ml liquid culture (centrifuge at 10000 g/ 30 s/ 4 °C)

from exponential P. tricornutum cells. After discarding the supernatant, the pellet was resuspended

in six ml NAES in six Eppendorf cups, each cup including 1 ml. At the same time, 100 beads and 1ml

water-saturated PCI was added into each cup. After vortex them 3x20 s, centrifuged for 10 min at

20000 g RT. Upper aqueous phase was moved into a new EP, 1 volume of water saturated PCI was

added into the aqueous phase, mixed gently and centrifuged 15 min, 20000 g RT again. The upper

aqueous phase was transferred to a new EP again and added 1 volume of chloroform, centrifuged 15

min, 20000 g RT. Subsequently, the aqueous supernatant was transferred to a new EP and added 0.7

volume of isopropanol to precipitate RNA, incubated the sample overnight at -20°C. At the next day,

centrifuged it full speed for 30 min and used 500 μL 70% EtOH to wash RNA. At last, the pellets were

dried in a desiccator and dissolved it in 15 μl of DEPC H2O.

Buffer B NAES Tris/HCl (pH 8.0) 0.1 M NaAc (pH 5,1) 50 mM Na2EDTA 0.02 M EDTA 10 mM NaCl 1.4 M SDS 1 % (w/v) 2-Mercaptoethanol 0.2 % (v/v)

5.2.4.3 cDNA synthesis via reverse transcription (RT)

Before cDNA synthesis, RNA isolation sample including potential genomic DNA had to be treated by

DNaseI in order to remove genomic DNA. Each 500 ng of RNA, 1 µl DNAseI (1 U/µl) and 2 µl 10x

reaction buffer were mixed in 20 µl total, and was incubated at RT for 20 min. After DNA removal,

RNA sample was checked on the agarose gel (selection). The cDNA synthesis was followed directly

with the SuperScriptTM II RT according to the manufacturer’s instructions. 1.3 µl random hexamer

primer and 1.7 µl DEPC H2O were added to the samples, and incubated at 70°C for 5 min. Then the

samples were kept on ice and 2 µl 10mM dNTPs and 4 µl reverse transcriptase buffer were added.

Following an incubation for 5 min at 25°C 1 µl reverse transciptase was added. After incubation for

60 min at 42°C the reaction was eventually stopped by incubating the samples at 70°C for 10 min.

5.2.4.4 Polymerase chain reaction (PCR)

The Phusion High Fidelity PCR Kit was used to amplify the DNA sequences from P. tricornutum gDNA

or cDNA. The concentration of the used primers (see supplementary material 7.3) was 5 pmol/µl and


72

the annealing temperature depended on the melting temperature of the primers. The reaction mix

consisted of the following components:

Phusion-HF-Buffer (4mM MgCl2) 5 μl

MgCl2 (50 mM) 2 μl

dNTPs (10 mM) 1 μl

Forward-Primer (5 pmol/µL) 1 μl

Reverse-Primer (5 pmol/µL) 1 μl

gDNA/cDNA solution (250 ng) 0.2 μl/2µl

Phusion High Fidelity DNA-Polymerase (5 U/µL)

0.25 μl

ad 25 µl ddH2O

Step Duration Temperature

1.Denaturation 1 min 98°C

2.Denaturation 20 s 98°C

3.Annealing 30 s Tm-3°C

4.Elongation Depends on sequence length 72°C

5.Final Elongation 4 min 72°C

6.Cool down 20 s 20°C

Step 2-4 were repeated 29 times (gDNA) or 40 times (cDNA).

Colony PCR was performed with Taq DNA Polymerase (Biotools) according to the manufacturer’s

instructions. A P. tricornutum colony, grown on selective medium after transfection, was

resuspended in 20 µl of ddH2O and boiled for 10 min at 95°C. After centrifugation (2000 g/ 10 min/

RT), the supernatant was used for colony PCR reaction. The reaction mix consisted of the following

components:

10xstandard Buffer with MgCl2 (Biotools) 2.5 µl

MgCl2 (50 mM) 1.25 µl

dNTPs (10 mM) 0.5 µl

For-Primer (5 pmol/µL) 0.5 µl

Rev-Primer (5 pmol/µL) 0.5 µl

Boiled colony suspension supernatant 5 µl

Taq- DNA-Polymerase (Biotools) 0.25 µl

ad 25 ul ddH2O

5.2.4.5 Agarose gel electrophoresis

For separation of DNA or RNA on an agarose gel, 10 µl sample and 2 µl loading buffer mix was

loaded onto a 1% to 2% agarose gel which was based on 1xTBE buffer. Roti®-GelStain (Roth) was


73

used to stain nucleic acid. For the elution of DNA fragments, the JetSorb DNA-Ectraction-Kit or PCR

purify kit was used according to manufacturer’s instructions.

DNA loading buffer (pH 7.0) 10xTBE Urea 4 M Tris/HCl (pH 8.8) 1 M EDTA 0.05 M Boric acid 0.83 M Sucrose 50% (w/v) EDTA 0.01 M Xylene cyanol 0.1 (w/v) Bromphenol blue 0.1 (w/v)

5.2.4.6 Sequencing

The sequencing of DNA was performed by the method described in Sanger, Nicklen et al. (Sanger,

Nicklen et al. 1977). The sequence reaction mixture was consisted of the following components:

Sequencing primer 1 µl ABI mix 2 µl Plasmid solution 4 µl ddH2O 3 µl

The following PCR reaction:

Step Duration Temperature

1.Denaturation 3 min 95°C

2.Denaturation 30 s 95°C

3.Annealing 30 s 50-55°C

4.Elongation 90 s 60°C

5.Final Elongation 4 min 60°C

6.Cool down 20 s 20°C

Step 2-4 were repeated 30 times.

After the PCR program, the reaction mix was moved to a new 1.5 ml EP and mixed with 64 µl 100%

ethanol and 26 µl ddH2O. The mix was saved in the dark for 30 min. After that, the mix was

centrifuged for 30 min at 2000 g, 4°C. The pellet was washed by 70% EtOH once, then was dried and

dissolved in 3 µl for amide loading dye. The ABI PRISM 377 DNA Sequencher was used to separate

the sequencing reaction. The nucleotides sequences were analyzed on Sequencher 5.1.)

5.2.4.7 Restriction and ligation

To prepare DNA fragment for ligation, normally, the mix is made up of the following components:

vector 0.3 µl

DNA 1 µl

restriction enzyme 1 0.25 µl


74

restriction enzyme 2 0.25 µl

10x SDB buffer 1 µl

ad 10 µl ddH2O

The restriction mix was incubated 30 min to 1h at 37°C. Then each sample was added 2ul loading

dye and loaded on agarose gel electrophoresis.

The eluted PCR products were ligated into pJet1.2/blunt cloning vector with the ClonJetTM PCT

Cloning Kit from Thermo Scientific/Fermentas. The ligation of DNA fragments into the expression

vector pPha-NR, the ratio of insert and vector was 3:1 and added 0.5-1 µl T4 ligase from Thermo

Scientific, and 1 µl 10xligase buffer in 10 µl volume. The ligation mix was incubated at RT for 30 min.

5.2.4.8 Transformation of E.coli

Before the transformation of E.coli, chemically RbCl-competent cells were prepared and stored at -

80°C. For the transformation of E.coli TOP10, 50 µl of RbCl-competent E. coli suspension were added

to the ligation mix and incubated on ice for 20-30 min. Then, the cells were heat shocked at 42°C for

45 s. After a short recovering time on ice the cells were plated on 1.5 % agar-LB plates containing 50

µg/ml ampicillin. The plate was incubated at 37°C overnight.

E. coli TOP10 cells were cultured overnight in 100 ml LB medium with streptomycin (50 µg/ml), a 1 L

culture with antibiotic was inoculated at 37°C, the starting OD600 is 0.1, at the same time, Sterile

MgCl2 and MgSO4 were added into the culture, a final concentration of them is 10 mM each. When

the cells concentration increased to OD600 of about 0.6, then centrifuged and used 33 ml RF1 to

resuspend it, incubated 30min on ice. After the second centrifugation, the pellet was resuspended in

50 ml RF2 and incubated 30 min on ice again. At last, aliquots of 100 µl were prepared in 4°C room

and frozen in liquid nitrogen and stored them at -80°C.

RF1 RF2

RbCl2 100 mM MOPS 10 mM

MnCl2x 4H2O 50 mM RbCl2 10 mM

KAc 30 mM CaCl2x2H2O 75 mM

CaCl2x2H2O 10 mM Glycerin 15% (w/v)

Glycerin 15% (w/v) Adjust pH to 5.8 with NaOH

Adjust pH to 5.8 with acetic acid

5.2.4.10 Transfection of P. tricornutum

Before transfection, the wild type P. tricornutum cells were cultured on f/2 agar plate (without

zeozin) for overnight. The concentration of cells on each plate is 108 cells in 100 μl f/2 liquid medium.

Normally, three plates’ wide type cells were prepared for one construct. The next day, the biolistic


75

transfection was carried out as described by Apt, Grossman et al. and Zaslavskaia, Lippmeier et al.

(Apt, Grossman et al. 1996, Zaslavskaia, Lippmeier et al. 2000). At the beginning, 50 µl M10 for each

construct, 5 µg plasmid (5000/plasmid concentration), 50 µl 2.5 M CaCl2 and 20 µl 0.1 M SP enzyme

was mixed using vortex for 1min. Then incubated 10 min at room temperature. After centrifugation

(full speed/ 5 min), the supernatant was threw away and then 250 µl 100% ETOH HPLC-Quality was

used to wash the pellet once. At last, 50 µl 100% ETOH HPLC-Quality was used to re-suspend the

pellet for transfection.

The transfection was carried out by using Biolistic PDS-1000/He Particle Delivery System. Before

transfection, a cleaning work was conducted with 100% ethanol (HPLC grade). After the cleaning, the

transfection was started. Firstly, 15 µl of the DNA bound microcarrier suspension was added on a

microcarrier membrane, then the components of the particle gun was assembled according to

manufacturer’s instructions. Secondly, when the vacuum increased to 25 psi, turn on the pressure

until to 1350 psi, then release immediately. Lastly, the transfected plates were sealed by Parafilm

and cultured at 22°C under the continuous light overnight. The next day, 1 ml f/2 medium was used

to wash each transfected plate to three f/2 ammonium agar plates including selective zeocin. The

plates were cultured under the continuous light at 22°C until colonies were visible.

5.2.4.11 Minicircles enrichment and isolation from A. carterae CCAM0512

A. carterae CCAM0512 was harvested from four to five weeks cultures by centrifugation (3000 g, 3

min). Alkaline extraction according to Bimboim and Doly for bacterial plasmid preparation was used

to enrich minicircles (Bimboim and Doly 1979). Additionally, different alkaline lysis based kits were

also used to enrich the minicircles. The following kits were used:

Company

PeqGOLD Plasmid Miniprep KitI Peq Lab

PureYieldTM Plasimid Miniprep System, 50 preps Promega

UltraClean 6 minute Mini Plasmid Prep Kit MoBio

High Yield Plasmid Mini Kit SLG

5.2.4.12 Transposon-insertion based approach

A transposon-insertion based approach was used to isolate individual minicircles from A. carterae

CCAM0512 in high copy number. The natural structure of plasmid-like minicircles provides a good

basis for transposon insertion and subsequent usage of bacterial proliferation. Firstly, a kanamycin

antibiotic resistance gene was inserted into the multi-cloning site of an EZ-Tn5 (Epicentre)

transposon in EZ-Tn5 pMOD-3 vector (EZ-Tn5TM pMODTM <R6Kγori/MCS> Transposon Construction


76

Vectors, EPICENTRE Company). EZ-Tn5 (Epicentre) transposon contains two mosaic ends (5’-

AGATGTGTATAAGAGACAG-3’), a multiple cloning site and R6Kγori of replication. R6Kγori-dependent

replication needs the pir gene product produced by TransforMax EC100D pir+ E.coli cells. Secondly,

the EZ-Tn5 (epicentre) transposon including the kanamycin antibiotic resistance gene was amplified

by transposon primers (see supplement 7.3). Subsequently, the transposon was inserted into the

target minicircles in vitro by using the reaction mixture (37°C, 2 hours) which is made up of the

following components:

Minicircles 6.5 µl/ 0.2 µg

Transposon 2 µl

EZ-Tn5 10xReaction buffer 1 µl

EZ-Tn5 Transposase 1 U/ µl 0.5 µl

ad 10 µl ddH2O

The 10xreaction buffer and transposase were provided by the EZ-Tn5TM Custom Transposome

Construction Kits. They were used according to the manufacturer’s instructions.

After transposon insertion reaction, as the EZ-Tn5 10xreaction buffer contains Mg2+, the reaction

mixture was not stopped by EZ-Tn5 10xstop solution as manufactors’ protocol. The reaction mixture

was purified by the next steps. Firstly, Phenol: Chloroform: Isoamyl Alcohol (25:24:1, v/v) was used

to purify the minicircles, then chloroform was used for the second step purification of transposon

inserted minicircles, and subsequently 2.5 volume 100% Ethanol and 0.1 volume NaAc, pH 4.8 was

used to the precipitation of transposon inserted minicircles. At last, electroporation procedures for

bacterial transformation (TransforMaxTM EC100DTM pir+ electrocompetent E.coli, 2.5 kV, fast charge

rate, 2 mm cuvette) was used. Each transformation mixture was cultured in 300 ml LB liquid medium

without antibiotic for 1 hour, and subsequently plated the culture on the plate. The plasmids

(potential individual minicircles) were isolated via standard plasmid preparation (Alkaline lysis). The

transposon insertion is schematically visualized in figure 5-1.


77

Fig. 5-1: Schematic depiction of transposon-insertion based isolation of minicircles.

It was shown that the minicircle provides a vector standard for the transposon insertion. The red region is the core region

of minicircles. The arrow region is the coding region of minicircles. The remainder of minicircles is called the non-coding

region. The transposon (marked by blue) contains an origin of replication and a kanamycin antibiotic resistence gene. The

transposon was inserted into the minicircles randomly in the reaction mix, so it was also possible inserted into the core

region, coding region and non-coding region.

5.2.4.13 DNA extraction, amplification, and sequencing of LSU rDNA domain D1-D6 and SSU rDNA

for A.carterae

Genomic DNA was isolated from 100 ml of exponentially growing A. carterae cells according to the

CTAB method described in Doyle and Doyle (Doyle and Doyle 1990). Extracted DNA was used as a

template, the Phusion High Fidelity PCR Kit was used to amplify approximately 1400 bp of the LSU

rDNA gene covering the variable domains D1-D6, using the primers (LSU-for:

ACCCGCTGAATTTAAGCATA; LSU-rev: CCACCATGCCCTCCTACTCA); and approximately 1700 bp of the

SSU rDNA gene, using the primers(SSU-for:GTCTCAAAGATTAAGCCATGCATGTC;SSU-

rev:CTTCTCCTTCCTCTAAGTGATAAGGTTC). Primers concentration is 5 pmol/µl and the applied

annealing temperature was calculated with the Tm calculator. Sequencing was done by Macrogen

Europe Company.

5.2.4.14 Sequence alignment and phylogenetic analyses

Sequences were aligned by clustalx2.0 and Mega 6.0 software as described in Tamura (Tamura,

Stecher et al. 2013). As outgroup species, the dinoflagellates Gonyaulax membranacea,

Gymnodinium dorsalisulcum, Heterocapsa arctica, Alexandrium fundyense were used. The data

matrix of LSU comprised 1183 aligned positions totally, which has excluded the hypervariable D2

domain region; for SSU data matrix, 1687 aligned positions were included. At the alignment, the

phylogenetic trees were constructed with maximum likelihood algorithm on the software of

Mega6.0. The optimal parameters were as follows: bootstrap replications: 1000; substitution model:


78

general time reversible model; ML heuristic method: nearest-neighbor-interchange (NNI); Branch

swap filter: very strong. All species included in the molecular analyses with their corresponding

GenBank accession numbers are shown at the behind of the name on the phylogenetic trees.

5.2.5 Protein analytics

5.2.5.1 Protein isolation from P. tricornutum

To isolate protein from P. tricornutum, two different methods were used. One is the alkaline lysis,

which can screen the expression of protein from small amount of culture samples. The other one is

using French press to extract protein for a subsequent fractionating.

Alkaline lysis: P. tricornutum cells were collected from 5-10 ml liquid medium by centrifugation at

1500 g/ 10 min/ 4°C. After discarding the supernatant, the pellet was resuspended in 1 ml ddH2O.

150 µl of lysis buffer was added to the pellet, vortexed and incubated on ice for 10 min for cell

lysis.French press passage: P. tricornutum cells were harvested by centrifugation at 1500 g/ 10 min/

4°C. From now on, every step was done on ice. After discarding the supernatant, the pellet was

resuspended in 2985 µl SolA buffer and 15 µl PIC was added into the suspension in a ratio of 1:200 in

order to inactivate proteases. Then the cells were disrupted through the pressure cell under a

constant pressure of 1000 psi. After the French press, centrifugation (1500 g/ 10 min/ 4°C) was

carried out to pellet the intact cells, while supernatant was used for the next experiments.

Lysis buffer Protease inhibitor cocktail (PIC)

NaOH 1.85 M Antipain 200 µg/mL

2-Mercaptoethanol* 7.5 % (v/v) Aprotinin 200 µg/mL

*2-Mercaptoethanol was added directly before an alkaline lysis was carried out.

Chymostatin 200 µg/mL

Elastatinal 200 µg/mL

Leupeptin 200 µg/mL

Pepstatin 200 µg/mL

Trypsin-Inhibitor 200 µg/mL

Na2EDTA 200 µg/mL

in 280 mM Hepes/KOH buffer (pH 7.9)

5.2.5.2 Protein extraction fractionation via carbonate extraction

Carbonate extraction was used to separate different kind of proteins, soluble, membrane associated

and integral membrane proteins from the whole protein extraction. The supernatant from the

French press was used to do carbonate extraction. Firstly, supernatant was centrifuged (120,000 g/

45 min/ 4°C) in order to pelletize the associated and integral membrane proteins, then the soluble

proteins (2 ml) in supernatant was transferred into two new Eppendorf cups and stored on ice. To


79

separate associated membrane proteins, the pellet was resuspended in carbonate buffer containing

PIC (1:200), incubated on ice for 30min and mixed gently by plastic head dropper. Subsequently, in

supernatant the associated membrane proteins was separated from integral membrane proteins by

centrifugation at 120000 g/ 45 min/ 4°C. Again, the associated membrane proteins in supernatant

was transferred into two new Eppendorf cups and stored on ice. While the integral membrane

proteins in pellet were resuspended in 2388 µl SolA buffer containing 12 µl PIC (1:200) and

transferred into another two new Eppendorf cups for the precipitation of proteins in next step.

Solubilization buffer A (SolA) (pH 7.5) Carbonate buffer (pH 11.5)

Imidazole 50 mM NaHCO3 100 mM

NaCl 50 mM EDTA 1 mM

6-aminohexanoic acid

2 mM

EDTA 1 mM

Sucrose 8.5 %

5.2.5.3 TCA protein precipitation

To precipitate proteins the TCA precipitation method was used. TCA was added to the samples to a

final concentration of 12.5%. After an incubation for 30 min on ice the samples were centrifuged

(20,000 x g/ 10 min/ 4°C). To remove residual TCA, the pellet was washed at least three times with

80% (v/v) acetone. After the last washing step the pellet was dried and dissolved in an appropriate

volume of urea loading buffer by incubated at 55°C for 10 min, then centrifuged for 30 s full speed

and save them in -20°C for next step analysis.

Urea loading buffer (pH 6.8)

Urea 8 M

Tris/Hcl 200 mM

EDTA 0.1 mM

SDS 5% (w/v)

Bromphenol blue 0.03% (w/v)

2-Mercaptoethanol* 1% (v/v)

*2-Mercaptoethanol was added to samples before using directly

5.2.5.4 Determination of protein concentration via Amido black

To determinate the concentration of proteins, Amido black method was used as described in Popov

(Popov, Schmitt et al. 1974). 3 µl protein samples were filled with 97 µl ddH2O to an end 100 µl.

Then the proteins were added 400 µl amido black staining solution. After mixing, the samples were

centrifuged at 20000 g/ 15 min/ 4°C. The protein pellet was washed by 500 µl washing solution and


80

centrifuged again. At last, the pellet was dried in dessicator and dissolved in 1 ml 200 mM NaOH.

After transferring sample to a cuvette, its absorption was measured at 615 nm wavelength by a

photometer. Before measuring, a blanking measurement was carried out by using 1 ml 200 mM

NaOH. Based on a standard curve, the calculation of the protein concentration was performed.

Amido black staining solution Amido black washing solution

Acetic acid 10 % (v/v) Methanol 90 % (v/v)

Methanol 90 % (v/v) Acetic acid 10 % (v/v)

a pinch of amido black

5.2.5.5 SDS-polyacrylamide gel electrophoresis (PAGE)

SDS-PAGE was used to separate proteins based on a discontinuous buffer system described by

Laemmli (Laemmli 1970).

Before loading onto the gel, the protein samples with urea loading dye were boiled up at 55°C for 10

min, the protein marker was PageRulerTM Prestained Protein Ladder (MBI Fermentas). The gel was

run at 140 V and 20 mA current in the stacking gel, 30 mA in the resolving gel,

4xStacking gel buffer 4xResolving gel buffer

Tris/HCl (pH 6.8) 500 mM Tris/HCl (pH 8.8) 1.5 M

SDS 0.4% (w/v) SDS 0.4% (w/v)

10XSDS running buffer

Tris 250 mM

Glycine 2 M

SDS 1% (w/v)

The SDS-PAGE gels consisted of the following components:

Resolving gel (12.5 %)* Resolving gel (12.5 %)*

Acrylamide 30 % (v/v) 4.1 mL 0.9 mL

dH2O 3.2 mL 2.8 mL

4x Resolving gel buffer 2.5 mL -

4x Stacking gel buffer - 1.25 mL

TEMED 20 µl 15 µl

APS 10 % (v/v) 150 µl 85 µl

*for separation of proteins with a molecular weight between 25-100 kDa.

5.2.5.6 Western blot analysis

Specific proteins separated by SDS-PAGE and was detected by Western blot. Firstly, the SDS-gel and

nitrocellulose was incubated on the transfer buffer for about 5 min. Whatman filter papers were


81

also incubated on the transfer buffer for about 1 min. Secondly, the semi-dry transfer system was

built up precisely from the direction of the anode to the cathode: three Whatman filter papers, the

blotting membrane (nitrocellulose the SDS-gel and then three Whatman filter papers. Protein

transfer was carried out at 50 V and an electric current of 1 mA/cm2 for 75 min.

After blotting, the membrane was transferred into a blocking bottle and incubated with blocking

solution for 1 h at RT to block unspecific binding sites. The membrane was incubated in the blocking

solution including the first antibody in an appropriate dilution over night at 4°C on a rotator. The

next day, the first antibody solution was removed and the membrane was washed thrice with TBS-T

buffer for 10 min. After the washing steps the membrane was incubated with the secondary

antibody for 1 h at RT. The membrane was washed once again thrice with TBS-T buffer and

eventually one time with TBS buffer.

The detection of the antibody-bound proteins was performed by the chemiluminescence, which

produced from the reaction catalyzed by the horseradish peroxidase when incubated with H2O2 and

the luminol containing ECL solution. Before the immunodetection, 30 % H2O2 was added to the ECL

solution at a ratio of 1:1000, which then was mixed and poured out on the top of membrane,

incubated for about 5 min. After incubation, the ECL solution was removed, the chemiluminescence

was detected by placing a photographic film on the membrane immediately.

Transfer buffer Blocking solution

Tris/HCl (pH 8.4) 25 mM Milk powder or BSA In TBS-T buffer

5 % (w/v)

Glycine 192 mM

Methanol 20 % (v/v)

TBS TBS-T

Tris/HCl (pH 7.5) 100 mM Tris/HCl (pH 7.5) 100 mM

NaCl 150 mM NaCl 150 mM

Tween 20 0.1 % (v/v)

ECL solution

Luminol 250 mM* 400 µl

Coumaric acid 90 mM*

178 µL

Tris/HCl 1 M (pH 8.5) 4 mL

ad 20 mL dH2O *in DMSO

5.2.5.7 Self-assembling GFP

The self-assembling GFP method was used to detect the topology of TIP4 protein in P. tricornutum.

In this approach GFP was split into two parts: long fragment GFPs1-10 and short fragment GFPs11.


82

For topology analyses of TIP4, GFPs11 was fused to the C-terminus of the protein, whereas the long

fragment GFPs1-10 was fused to the C-terminus of specific markers for the ER lumen (PDI protein)

and the PPC marker (Hsp70BTS), respectively. Only when both parts are targeted to the same

cellular compartment can assemble to produce green fluorescence.

After transfection, the colonies were checked by colony PCR via amplification of the first cloning site

(SpeI/SacII) with an NR promoter primer and an internal primer from the compartment marker

genes.

5.2.5.8 Construction of eGFP fusion proteins

All the C-terminus of predicted protein were fused to eGFP or mRFP and cloned into pPha-NR vector

(GenBank accession no. JN180663) or pPha-dual-NR vectors, transfected into the diatom P.

tricornutum and expressed via the nitrate reductase promoter. The sequences of genes containing

introns or predicted gene model was not confirmed by EST data were amplified from cDNA, the rest

of genes were amplified from gDNA. For further information about the protein sequences and

primer sequences used for transfection and PCR, respectively, see the supplemental material.

Biolistic transfection was used as described by Sommer (Sommer, Gould et al. 2007).

5.2.5.9 Confocal laser scanning microscopy

All P. tricornutum transfectants were analyzed with a confocal laser scanning microscope. Leica TCS

SP2 using a HCX PL APO 40× /1.25 to 0.75 oil CS objective after fixing with 4% paraformaldehyde–

0.0075% glutaraldehyde in 1× phosphate buffered saline buffer. The fluorescence of enhanced green

fluorescent protein (eGFP) and plastid autofluorescence was excited with an argon laser at 488 nm

and detected with two photomultiplier tubes at a bandwidths of 500 to 520 nm and 625 to 720 nm

for eGFP and plastid autofluorescence, respectively. The fluorescence of mRFP was excited with a

HeNe 1.2 mW laser at 543 nm and detected with a photomultiplier tubes at a bandwidths of 580 to

600 nm for mRFP.

5.2.5.10 Electron microscopy

For transmission electron microscopic analyses P. tricornutum cells were harvested (5 min, 2000 g),

high pressure frozen (Wohlwend HPF Compact 02) and freeze-substituted (Leica AFS2) with a

medium on acetone basis containing 0.25% osmium tetroxide, 0.2% uranyl acetate and 5% water.

Samples were then embedded in Epon 812 resin (Fluka) and cut to 50 nm ultrathin sections. Details

of the procedure are described in Peschke et al. For immunolabeling on ultrathin sections a primary


83

antibody against GFP (Rockland) (dilution 1:500 and 1:1000) and a secondary antibody coupled to

ultrasmall gold particles (Aurion) (dilution 1:100) were used, followed by silver enhancement. The

procedure of immunolabeling and silver enhancement are described in Rachel and Danscher,

respectively (Danscher 1981, Rachel, Meyer et al. 2010). After silver enhancement sections were

post-stained with 2% uranyl acetate and 0.5% lead citrate. Samples were analysed using a JEOL 2100

electron microscope.

References

84

6 References

Adl, S. M., A. G. Simpson, M. A. Farmer, R. A. Andersen, O. R. Anderson, J. R. Barta, S. S. Bowser, G.

Brugerolle, R. A. Fensome, S. Fredericq, T. Y. James, S. Karpov, P. Kugrens, J. Krug, C. E. Lane, L. A.

Lewis, J. Lodge, D. H. Lynn, D. G. Mann, R. M. McCourt, L. Mendoza, O. Moestrup, S. E. Mozley-

Standridge, T. A. Nerad, C. A. Shearer, A. V. Smirnov, F. W. Spiegel and M. F. Taylor (2005). "The

new higher level classification of eukaryotes with emphasis on the taxonomy of protists." J

Eukaryot Microbiol 52(5): 399-451.

Ahmed, S. U., E. Rojo, V. Kovaleva, S. Venkataraman, J. E. Dombrowski, K. Matsuoka and N. V.

Raikhel (2000). "The plant vacuolar sorting receptor AtELP is involved in transport of NH2-terminal

propeptide-containing vacuolar proteins in Arabidopsis thaliana." The Journal of cell biology

149(7): 1335-1344.

Apt, K. E., A. Grossman and P. Kroth-Pancic (1996). "Stable nuclear transformation of the diatom

Phaeodactylum tricornutum." Molecular and General Genetics MGG 252(5): 572-579.

Archibald, J. M. (2007). "Nucleomorph genomes: structure, function, origin and evolution."

Bioessays 29(4): 392-402.

Arighi, C. N., L. M. Hartnell, R. C. Aguilar, C. R. Haft and J. S. Bonifacino (2004). "Role of the

mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor." The

Journal of cell biology 165(1): 123-133.

Aviezer-Hagai, K., H. Nelson and N. Nelson (2000). "Cloning and expression of cDNAs encoding plant

V-ATPase subunits in the corresponding yeast null mutants." Biochimica et Biophysica Acta (BBA)-

Bioenergetics 1459(2): 489-498.

Baïet, B., C. Burel, B. Saint-Jean, R. Louvet, L. Menu-Bouaouiche, M.-C. Kiefer-Meyer, E. Mathieu-

Rivet, T. Lefebvre, H. Castel and A. Carlier (2011). "N-glycans of Phaeodactylum tricornutum

diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme." Journal

of Biological Chemistry 286(8): 6152-6164.

Bachvaroff, T. R., G. T. Concepcion, C. R. Rogers, E. M. Herman and C. F. Delwiche (2004).

"Dinoflagellate expressed sequence tag data indicate massive transfer of chloroplast genes to the

nuclear genome." Protist 155(1): 65-78.

Bachvaroff, T. R., M. V. Sanchez-Puerta and C. F. Delwiche (2006). "Rate variation as a function of

gene origin in plastid-derived genes of peridinin-containing dinoflagellates." J Mol Evol 62(1): 42-

52.

Baiet, B., C. Burel, B. Saint-Jean, R. Louvet, L. Menu-Bouaouiche, M. C. Kiefer-Meyer, E. Mathieu-

Rivet, T. Lefebvre, H. Castel, A. Carlier, J. P. Cadoret, P. Lerouge and M. Bardor (2011). "N-glycans

References

85

of Phaeodactylum tricornutum diatom and functional characterization of its N-

acetylglucosaminyltransferase I enzyme." J Biol Chem 286(8): 6152-6164.

Baiges, I., A. R. Schaffner, M. J. Affenzeller and A. Mas (2002). "Plant aquaporins." Physiol Plant

115(2): 175-182.

Balciunas, D. and S. C. Ekker (2005). "Trapping fish genes with transposons." Zebrafish 1(4): 335-341.

Barbrook, A., H. Symington, R. Nisbet, A. Larkum and C. Howe (2001). "Organisation and expression

of the plastid genome of the dinoflagellate Amphidinium operculatum." Molecular Genetics and

Genomics 266(4): 632-638.

Barbrook, A. C., R. G. Dorrell, J. Burrows, L. J. Plenderleith, R. E. Nisbet and C. J. Howe (2012).

"Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of

the dinoflagellate Amphidinium carterae." Plant Mol Biol 79(4-5): 347-357.

Barbrook, A. C. and C. J. Howe (2000). "Minicircular plastid DNA in the dinoflagellate Amphidinium

operculatum." Mol Gen Genet 263(1): 152-158.

Barbrook, A. C., N. Santucci, L. J. Plenderleith, R. G. Hiller and C. J. Howe (2006). "Comparative

analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes." BMC Genomics 7:

297.

Barlowe, C. (2003). "Signals for COPII-dependent export from the ER: what's the ticket out?" Trends

in cell biology 13(6): 295-300.

Bimboim, H. and J. Doly (1979). "A rapid alkaline extraction procedure for screening recombinant

plasmid DNA." Nucleic acids research 7(6): 1513-1523.

Boczar, B. A., J. Liston and R. A. Cattolico (1991). "Characterization of satellite DNA from three

marine dinoflagellates (dinophyceae): Glenodinium sp. and two members of the toxic genus,

Protogonyaulax." Plant physiology 97(2): 613-618.

Bodansky, S., L. B. Mintz and D. S. Holmes (1979). "The mesokaryote Gyrodiniumcohnii lacks

nucleosomes." Biochemical and biophysical research communications 88(4): 1329-1336.

Bodyl, A., P. Mackiewicz and P. Gagat (2012). "Organelle evolution: Paulinella breaks a paradigm."

Curr Biol 22(9): R304-306.

Bodył, A., P. Mackiewicz and J. Stiller (2010). "Comparative genomic studies suggest that the

cyanobacterial endosymbionts of the amoeba Paulinella chromatophora possess an import

apparatus for nuclear‐encoded proteins." Plant Biology 12(4): 639-649.

Boevink, P., K. Oparka, S. S. Cruz, B. Martin, A. Betteridge and C. Hawes (1998). "Stacks on tracks: the

plant Golgi apparatus traffics on an actin/ER network†." The Plant Journal 15(3): 441-447.

Bolte, K., L. Bullmann, F. Hempel, A. Bozarth, S. Zauner and U. G. MAIER (2009). "Protein Targeting

into Secondary Plastids1." Journal of Eukaryotic Microbiology 56(1): 9-15.

References

86

Bolte, S., S. Brown and B. Satiat-Jeunemaitre (2004). "The N-myristoylated Rab-GTPase m-Rabmc is

involved in post-Golgi trafficking events to the lytic vacuole in plant cells." Journal of cell science

117(6): 943-954.

Bondili, J. S., A. Castilho, L. Mach, J. Glössl, H. Steinkellner, F. Altmann and R. Strasser (2006).

"Molecular cloning and heterologous expression of β1, 2-xylosyltransferase and core α1, 3-

fucosyltransferase from maize." Phytochemistry 67(20): 2215-2224.

Bonifacino, J. S. and J. H. Hurley (2008). "Retromer." Current opinion in cell biology 20(4): 427-436.

Bonifacino, J. S. and L. M. Traub (2003). "Signals for sorting of transmembrane proteins to

endosomes and lysosomes." Annu Rev Biochem 72: 395-447.

Bosch, D., A. Castilho, A. Loos, A. Schots and H. Steinkellner (2013). "N-Glycosylation of Plant-

produced Recombinant Proteins." Current Pharmaceutical Design 19(31): 5503-5512.

Bouligand, Y. and V. Norris (2001). "Chromosome separation and segregation in dinoflagellates

andbacteria may depend on liquid crystalline states." Biochimie 83(2): 187-192.

Boursiac, Y., S. Chen, D.-T. Luu, M. Sorieul, N. van den Dries and C. Maurel (2005). "Early effects of

salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin

expression." Plant physiology 139(2): 790-805.

Breton, C., J. Mucha and C. Jeanneau (2001). "Structural and functional features of

glycosyltransferases." Biochimie 83(8): 713-718.

Burki, F., B. Imanian, E. Hehenberger, Y. Hirakawa, S. Maruyama and P. J. Keeling (2014).

"Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates." Eukaryot Cell 13(2):

246-255.

Burki, F., Y. Inagaki, J. Brate, J. M. Archibald, P. J. Keeling, T. Cavalier-Smith, M. Sakaguchi, T.

Hashimoto, A. Horak, S. Kumar, D. Klaveness, K. S. Jakobsen, J. Pawlowski and K. Shalchian-Tabrizi

(2009). "Large-scale phylogenomic analyses reveal that two enigmatic protist lineages, telonemia

and centroheliozoa, are related to photosynthetic chromalveolates." Genome Biol Evol 1: 231-238.

Carlson, C. M., J. L. Frandsen, N. Kirchhof, R. S. McIvor and D. A. Largaespada (2005). "Somatic

integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor

development in the mouse." Proceedings of the National Academy of Sciences of the United

States of America 102(47): 17059-17064.

Carlton, J., M. Bujny, B. J. Peter, V. M. Oorschot, A. Rutherford, H. Mellor, J. Klumperman, H. T.

McMahon and P. J. Cullen (2004). "Sorting nexin-1 mediates tubular endosome-to-TGN transport

through coincidence sensing of high-curvature membranes and 3-phosphoinositides." Current

biology 14(20): 1791-1800.

Cavalier-Smith, T. (1998). "A revised six-kingdom system of life." Biol Rev Camb Philos Soc 73(3):

203-266.

References

87

Cavalier-Smith, T. (1999). "Principles of Protein and Lipid Targeting in Secondary Symbiogenesis:

Euglenoid, Dinoflagellate, and Sporozoan Plastid Origins and the Eukaryote Family Tree1, 2."

Journal of Eukaryotic Microbiology 46(4): 347-366.

Cavalier-Smith, T. (2000). "membrane heredity and early chloroplast evolution." trends in plant

science 5: 1360-1385.

Cavalier-Smith, T. (2002). "The phagotrophic origin of eukaryotes and phylogenetic classification of

protozoa." international journal of systematic and evolutionary microbiology 52: 297-354.

Cavalier-Smith, T. and E. E.-Y. Chao (2003). "Phylogeny and classification of phylum Cercozoa

(Protozoa)." Protist 154(3): 341-358.

Chaumont, F., F. Barrieu, E. Wojcik, M. J. Chrispeels and R. Jung (2001). "Aquaporins constitute a

large and highly divergent protein family in maize." Plant Physiology 125(3): 1206-1215.

Chaumont, F. and S. D. Tyerman (2014). "Aquaporins: highly regulated channels controlling plant

water relations." Plant Physiol 164(4): 1600-1618.

Chevalier, A. S. and F. Chaumont (2014). "Trafficking of Plant Plasma Membrane Aquaporins:

Multiple Regulation Levels and Complex Sorting Signals." Plant Cell Physiol.

Chow, M. H., K. T. Yan, M. J. Bennett and J. T. Wong (2010). "Birefringence and DNA condensation of

liquid crystalline chromosomes." Eukaryotic cell 9(10): 1577-1587.

Clarke, M., J. Köhler, Q. Arana, T. Liu, J. Heuser and G. Gerisch (2002). "Dynamics of the vacuolar H+-

ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells."

Journal of cell science 115(14): 2893-2905.

Contreras, I., E. Ortiz-Zapater, L. M. Castilho and F. Aniento (2000). "Characterization of Cop I coat

proteins in plant cells." Biochem Biophys Res Commun 273(1): 176-182.

Cullen, P. J. and H. C. Korswagen (2012). "Sorting nexins provide diversity for retromer-dependent

trafficking events." Nature Cell Biology 14(1): 29-37.

Dagan, T. and W. Martin (2009). "Getting a better picture of microbial evolution en route to a

network of genomes." Philos Trans R Soc Lond B Biol Sci 364(1527): 2187-2196.

Dang, Y. and B. R. Green (2009). "Substitutional editing of Heterocapsa triquetra chloroplast

transcripts and a folding model for its divergent chloroplast 16S rRNA." Gene 442(1-2): 73-80.

Dang, Y. and B. R. Green (2010). "Long transcripts from dinoflagellate chloroplast minicircles suggest

"rolling circle" transcription." J Biol Chem 285(8): 5196-5203.

Danscher, G. (1981). "Localization of gold in biological tissue." Histochemistry 71(1): 81-88.

daSilva, L. L., E. L. Snapp, J. Denecke, J. Lippincott-Schwartz, C. Hawes and F. Brandizzi (2004).

"Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in

plant cells." Plant Cell 16(7): 1753-1771.

References

88

De Marchis, F., M. Bellucci and A. Pompa (2013). "Traffic of human alpha-mannosidase in plant cells

suggests the presence of a new endoplasmic reticulum-to-vacuole pathway without involving the

Golgi complex." Plant Physiol 161(4): 1769-1782.

Dettmer, J., A. Hong-Hermesdorf, Y.-D. Stierhof and K. Schumacher (2006). "Vacuolar H+-ATPase

activity is required for endocytic and secretory trafficking in Arabidopsis." The Plant Cell 18(3):

715-730.

Dettmer, J., T. Y. Liu and K. Schumacher (2010). "Functional analysis of Arabidopsis V-ATPase subunit

VHA-E isoforms." Eur J Cell Biol 89(2-3): 152-156.

Deusch, O., G. Landan, M. Roettger, N. Gruenheit, K. V. Kowallik, J. F. Allen, W. Martin and T. Dagan

(2008). "Genes of cyanobacterial origin in plant nuclear genomes point to a heterocyst-forming

plastid ancestor." Mol Biol Evol 25(4): 748-761.

Dodge, J. and J. Lee (2000). "Phylum dinoflagellata." Illustrated Guide to the Protozoa. Society of

Protozoologists. Lawrence, KA: 656-689.

Dorrell, R. G. and A. G. Smith (2011). "Do red and green make brown?: perspectives on plastid

acquisitions within chromalveolates." Eukaryot Cell 10(7): 856-868.

Doyle, J. and J. Doyle (1990). "Isolation of DNA from small amounts of plant tissues." BRL focus 12:

13-15.

Ebine, K., T. Inoue, J. Ito, E. Ito, T. Uemura, T. Goh, H. Abe, K. Sato, A. Nakano and T. Ueda (2014).

"Plant vacuolar trafficking occurs through distinctly regulated pathways." Curr Biol 24(12): 1375-

1382.

Endler, A., S. Meyer, S. Schelbert, T. Schneider, W. Weschke, S. W. Peters, F. Keller, S. Baginsky, E.

Martinoia and U. G. Schmidt (2006). "Identification of a vacuolar sucrose transporter in barley and

Arabidopsis mesophyll cells by a tonoplast proteomic approach." Plant Physiology 141(1): 196-207.

Facchinelli, F., M. Pribil, U. Oster, N. J. Ebert, D. Bhattacharya, D. Leister and A. P. Weber (2013).

"Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in

plastid endosymbiosis." Planta 237(2): 637-651.

Fast, N. M., J. C. Kissinger, D. S. Roos and P. J. Keeling (2001). "Nuclear-encoded, plastid-targeted

genes suggest a single common origin for apicomplexan and dinoflagellate plastids." Molecular

Biology and Evolution 18(3): 418-426.

Fischer, M. and R. Kaldenhoff (2008). "On the pH regulation of plant aquaporins." J Biol Chem

283(49): 33889-33892.

Fitchette‐Lainé, A. C., V. Gomord, A. Chekkafi and L. Faye (1994). "Distribution of xylosylation and

fucosylation in the plant Golgi apparatus." The Plant Journal 5(5): 673-682.

Frigerio, L., G. Hinz and D. G. Robinson (2008). "Multiple vacuoles in plant cells: rule or exception?"

Traffic 9(10): 1564-1570.

References

89

Frigerio, L., N. A. Jolliffe, A. Di Cola, D. H. Felipe, N. Paris, J.-M. Neuhaus, J. M. Lord, A. Ceriotti and L.

M. Roberts (2001). "The internal propeptide of the ricin precursor carries a sequence-specific

determinant for vacuolar sorting." Plant physiology 126(1): 167-175.

Frommolt, R., S. Werner, H. Paulsen, R. Goss, C. Wilhelm, S. Zauner, U. G. Maier, A. R. Grossman, D.

Bhattacharya and M. Lohr (2008). "Ancient recruitment by chromists of green algal genes

encoding enzymes for carotenoid biosynthesis." Mol Biol Evol 25(12): 2653-2667.

Gabrielsen, T. M., M. A. Minge, M. Espelund, A. Tooming-Klunderud, V. Patil, A. J. Nederbragt, C. Otis,

M. Turmel, K. Shalchian-Tabrizi, C. Lemieux and K. S. Jakobsen (2011). "Genome evolution of a

tertiary dinoflagellate plastid." PLoS One 6(4): e19132.

Galili, A. V. a. G. (2001). "The endomembrane system and the problem of protein sorting." plant

Physiol 125: 115-118.

Gattolin, S., M. Sorieul and L. Frigerio (2011). "Mapping of tonoplast intrinsic proteins in maturing

and germinating Arabidopsis seeds reveals dual localization of embryonic TIPs to the tonoplast

and plasma membrane." Mol Plant 4(1): 180-189.

Gattolin, S., M. Sorieul, P. R. Hunter, R. H. Khonsari and L. Frigerio (2009). "In vivo imaging of the

tonoplast intrinsic protein family in Arabidopsis roots." BMC Plant Biol 9: 133.

Gautier, A., L. Michel-Salamin, E. Tosi-Couture, A. McDowall and J. Dubochet (1986). "Electron

microscopy of the chromosomes of dinoflagellates in situ: confirmation of Bouligand's liquid

crystal hypothesis." Journal of ultrastructure and molecular structure research 97(1): 10-30.

Gautreau, A., K. Oguievetskaia and C. Ungermann (2014). "Function and regulation of the endosomal

fusion and fission machineries." Cold Spring Harb Perspect Biol 6(3).

Geldner, N., N. Anders, H. Wolters, J. Keicher, W. Kornberger, P. Muller, A. Delbarre, T. Ueda, A.

Nakano and G. Jürgens (2003). "The Arabidopsis GNOM ARF-GEF mediates endosomal recycling,

auxin transport, and auxin-dependent plant growth." Cell 112(2): 219-230.

Geuze, W. H. a. H. J. (1995). "Intracellular trafficking of lysosomal membrane proteins." Bioessays 18:

379-389.

Gibbs, S. P. (1979). "The route of entry of cytoplasmically synthesized proteins into chloroplasts of

algae possessing chloroplast ER." Journal of cell science 35(1): 253-266.

Gillespie, J., S. W. Rogers, M. Deery, P. Dupree and J. C. Rogers (2005). "A unique family of proteins

associated with internalized membranes in protein storage vacuoles of the Brassicaceae." The

Plant Journal 41(3): 429-441.

Gould, S. B., U. G. Maier and W. F. Martin (2015). "Protein Import and the Origin of Red Complex

Plastids." Curr Biol 25(12): R515-R521.

Gould, S. B., R. F. Waller and G. I. McFadden (2008). "Plastid evolution." Annu. Rev. Plant Biol. 59:

491-517.

References

90

Green, B. R. (2004). "The chloroplast genome of dinoflagellates–a reduced instruction set?" Protist

155(1): 23-31.

Green, B. R. (2011). "After the primary endosymbiosis: an update on the chromalveolate hypothesis

and the origins of algae with Chl c." Photosynth Res 107(1): 103-115.

Grosche, C. (2012). "Spezielle Leistungen der plastide: RNA-edierung in Landpflanzen,

Genomreduktion und Proteinimport in Peridinin-haltigen Dinoflagellaten." Dissertation.

Grosche, C., H. T. Funk, U. G. Maier and S. Zauner (2012). "The chloroplast genome of Pellia

endiviifolia: gene content, RNA-editing pattern, and the origin of chloroplast editing." Genome

Biol Evol 4(12): 1349-1357.

Grosche, C., F. Hempel, K. Bolte, S. Zauner and U. G. Maier (2014). "The periplastidal compartment: a

naturally minimized eukaryotic cytoplasm." Curr Opin Microbiol 22: 88-93.

Gruber, A., G. Rocap, P. G. Kroth, E. V. Armbrust and T. Mock (2015). "Plastid proteome prediction

for diatoms and other algae with secondary plastids of the red lineage." Plant J 81(3): 519-528.

Gruber, A., S. Vugrinec, F. Hempel, S. B. Gould, U. G. Maier and P. G. Kroth (2007). "Protein targeting

into complex diatom plastids: functional characterisation of a specific targeting motif." Plant Mol

Biol 64(5): 519-530.

Höfte, H., L. Hubbard, J. Reizer, D. Ludevid, E. M. Herman and M. J. Chrispeels (1992). "Vegetative

and seed-specific forms of tonoplast intrinsic protein in the vacuolar membrane of Arabidopsis

thaliana." Plant Physiology 99(2): 561-570.

Hackett, J. D., H. S. Yoon, S. Li, A. Reyes-Prieto, S. E. Rummele and D. Bhattacharya (2007).

"Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the

association of rhizaria with chromalveolates." Mol Biol Evol 24(8): 1702-1713.

Hackett, J. D., H. S. Yoon, M. B. Soares, M. F. Bonaldo, T. L. Casavant, T. E. Scheetz, T. Nosenko and D.

Bhattacharya (2004). "Migration of the plastid genome to the nucleus in a peridinin

dinoflagellate." Curr Biol 14(3): 213-218.

Hackett, P. B., D. A. Largaespada and L. J. Cooper (2010). "A transposon and transposase system for

human application." Molecular Therapy 18(4): 674-683.

Hampl, V., L. Hug, J. W. Leigh, J. B. Dacks, B. F. Lang, A. G. Simpson and A. J. Roger (2009).

"Phylogenomic analyses support the monophyly of Excavata and resolve relationships among

eukaryotic "supergroups"." Proc Natl Acad Sci U S A 106(10): 3859-3864.

Hanton, S. L., L. Renna, L. E. Bortolotti, L. Chatre, G. Stefano and F. Brandizzi (2005). "Diacidic motifs

influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells."

Plant Cell 17(11): 3081-3093.

References

91

Happel, N., S. Höning, J. M. Neuhaus, N. Paris, D. G. Robinson and S. E. Holstein (2004).

"ArabidopsisµA‐adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR‐

PS1." The Plant Journal 37(5): 678-693.

Hara-Nishimura, I., R. Matsushima, T. Shimada and M. Nishimura (2004). "Diversity and formation of

endoplasmic reticulum-derived compartments in plants. Are these compartments specific to plant

cells?" Plant Physiol 136(3): 3435-3439.

Hara‐Hishimura, I., Y. Takeuchi, K. Inoue and M. Nishimura (1993). "Vesicle transport and processing

of the precursor to 2S albumin in pumpkin." The Plant Journal 4(5): 793-800.

Harbour, M. E., S. Y. Breusegem, R. Antrobus, C. Freeman, E. Reid and M. N. Seaman (2010). "The

cargo-selective retromer complex is a recruiting hub for protein complexes that regulate

endosomal tubule dynamics." Journal of cell science 123(21): 3703-3717.

Harper, J. T. and P. J. Keeling (2003). "Nucleus-encoded, plastid-targeted glyceraldehyde-3-

phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids." Mol Biol

Evol 20(10): 1730-1735.

Hegedus, D. D., C. Coutu, M. Harrington, B. Hope, K. Gerbrandt and I. Nikolov (2015). "Multiple

internal sorting determinants can contribute to the trafficking of cruciferin to protein storage

vacuoles." Plant molecular biology 88(1-2): 3-20.

Hempel, F., L. Bullmann, J. Lau, S. Zauner and U. G. Maier (2009). "ERAD-derived preprotein

transport across the second outermost plastid membrane of diatoms." Molecular biology and

evolution 26(8): 1781-1790.

Hempel, F., G. Felsner and U. G. Maier (2010). "New mechanistic insights into pre-protein transport

across the second outermost plastid membrane of diatoms." Mol Microbiol 76(3): 793-801.

Herman, E. and M. Schmidt (2004). "Endoplasmic reticulum to vacuole trafficking of endoplasmic

reticulum bodies provides an alternate pathway for protein transfer to the vacuole." Plant Physiol

136(3): 3440-3446.

Herman, E. M. (2008). "Endoplasmic reticulum bodies: solving the insoluble." Current opinion in

plant biology 11(6): 672-679.

Hiller, R. G. (2001). "'Empty' minicircles and petB/atpA and psbD/psbE (cytb559 alpha) genes in

tandem in Amphidinium carterae plastid DNA." FEBS Lett 505(3): 449-452.

Hiller, R. G. (2001). "‘Empty’minicircles and petB/atpA and psbD/psbE (cytb 559 α) genes in tandem

in Amphidinium carterae plastid DNA." FEBS letters 505(3): 449-452.

Hohl, I., D. G. Robinson, M. J. Chrispeels and G. Hinz (1996). "Transport of storage proteins to the

vacuole is mediated by vesicles without a clathrin coat." Journal of Cell Science 109(10): 2539-

2550.

Holstein, S. E. (2002). "Clathrin and plant endocytosis." Traffic 3(9): 614-620.

References

92

Horazdovsky, B., B. Davies, M. Seaman, S. McLaughlin, S.-H. Yoon and S. Emr (1997). "A sorting

nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the

vacuolar protein-sorting receptor." Molecular biology of the cell 8(8): 1529-1541.

Howe, C. J., A. C. Barbrook, V. L. Koumandou, R. E. Nisbet, H. A. Symington and T. F. Wightman

(2003). "Evolution of the chloroplast genome." Philos Trans R Soc Lond B Biol Sci 358(1429): 99-

106; discussion 106-107.

Howe, C. J., R. E. Nisbet and A. C. Barbrook (2008). "The remarkable chloroplast genome of

dinoflagellates." J Exp Bot 59(5): 1035-1045.

Hunter, P. R., C. P. Craddock, S. Di Benedetto, L. M. Roberts and L. Frigerio (2007). "Fluorescent

reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar

compartment in Arabidopsis cells." Plant Physiol 145(4): 1371-1382.

Hwang, I. (2008). "Sorting and anterograde trafficking at the Golgi apparatus." Plant Physiol 148(2):

673-683.

Islam, M. S., N. I. A. Patwary, N. H. Muzahid, S. M. Shahik, M. Sohel and M. A. Hasan (2014). "A

systematic study on structure and function of ATPase of wuchereria bancrofti." Toxicology

international 21(3): 269.

Jürgens, G. (2004). "Membrane trafficking in plants." Annu. Rev. Cell Dev. Biol. 20: 481-504.

Jaillais, Y., M. Santambrogio, F. Rozier, I. Fobis-Loisy, C. Miège and T. Gaude (2007). "The retromer

protein VPS29 links cell polarity and organ initiation in plants." Cell 130(6): 1057-1070.

Jauh, G.-Y., T. E. Phillips and J. C. Rogers (1999). "Tonoplast intrinsic protein isoforms as markers for

vacuolar functions." The Plant Cell 11(10): 1867-1882.

Jiang, L., T. E. Phillips, S. W. Rogers and J. C. Rogers (2000). "Biogenesis of the protein storage

vacuole crystalloid." The Journal of cell biology 150(4): 755-770.

Jin, J. B., Y. A. Kim, S. J. Kim, S. H. Lee, D. H. Kim, G.-W. Cheong and I. Hwang (2001). "A new

dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central

vacuole in Arabidopsis." The Plant Cell 13(7): 1511-1526.

Johanson, U. and S. Gustavsson (2002). "A new subfamily of major intrinsic proteins in plants."

Molecular biology and evolution 19(4): 456-461.

Johanson, U., M. Karlsson, I. Johansson, S. Gustavsson, S. Sjövall, L. Fraysse, A. R. Weig and P.

Kjellbom (2001). "The complete set of genes encoding major intrinsic proteins in Arabidopsis

provides a framework for a new nomenclature for major intrinsic proteins in plants." Plant

physiology 126(4): 1358-1369.

Johansson, I., M. Karlsson, U. Johanson, C. Larsson and P. Kjellbom (2000). "The role of aquaporins in

cellular and whole plant water balance." Biochim Biophys Acta 1465(1-2): 324-342.

References

93

Johnson, A. E. and M. A. van Waes (1999). "The translocon: a dynamic gateway at the ER

membrane." Annual review of cell and developmental biology 15(1): 799-842.

Johnson, K. D. and M. J. Chrispeels (1992). "Tonoplast-bound protein kinase phosphorylates

tonoplast intrinsic protein." Plant Physiology 100(4): 1787-1795.

Jolliffe, N., C. Craddock and L. Frigerio (2005). "Pathways for protein transport to seed storage

vacuoles." Biochemical Society Transactions 33(Pt 5): 1016-1018.

Jolliffe, N. A., J. C. Brown, U. Neumann, M. Vicre, A. Bachi, C. Hawes, A. Ceriotti, L. M. Roberts and L.

Frigerio (2004). "Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus

communis endosperm involves the Golgi and VSR-like receptors." Plant J 39(6): 821-833.

Kajiura, H., T. Okamoto, R. Misaki, Y. Matsuura and K. Fujiyama (2012). "Arabidopsis beta1,2-

xylosyltransferase: substrate specificity and participation in the plant-specific N-glycosylation

pathway." J Biosci Bioeng 113(1): 48-54.

Kaldenhoff, R., A. Bertl, B. Otto, M. Moshelion and N. Uehlein (2007). "Characterization of plant

aquaporins." Methods Enzymol 428: 505-531.

Kaldenhoff, R. and M. Fischer (2006). "Aquaporins in plants." Acta Physiol (Oxf) 187(1-2): 169-176.

Kaldenhoff, R., M. Ribas-Carbo, J. F. Sans, C. Lovisolo, M. Heckwolf and N. Uehlein (2008).

"Aquaporins and plant water balance." Plant Cell Environ 31(5): 658-666.

Kalthoff, C., S. Groos, R. Kohl, S. Mahrhold and E. J. Ungewickell (2002). "Clint: a novel clathrin-

binding ENTH-domain protein at the Golgi." Mol Biol Cell 13(11): 4060-4073.

Karlsson, M., I. Johansson, M. Bush, M. C. McCann, C. Maurel, C. Larsson and P. Kjellbom (2000). "An

abundant TIP expressed in mature highly vacuolated cells." The Plant Journal 21(1): 83-90.

Keeling, P. J. (2009). "Chromalveolates and the evolution of plastids by secondary endosymbiosis." J

Eukaryot Microbiol 56(1): 1-8.

Keeling, P. J. (2010). "The endosymbiotic origin, diversification and fate of plastids." Philos Trans R

Soc Lond B Biol Sci 365(1541): 729-748.

Keeling, P. J. (2013). "The number, speed, and impact of plastid endosymbioses in eukaryotic

evolution." Annual review of plant biology 64: 583-607.

Kim, H. S., J. H. Jeon, K. J. Lee and K. Ko (2014). "N-glycosylation modification of plant-derived virus-

like particles: an application in vaccines." Biomed Res Int 2014: 249519.

Kim, J. M., S. Vanguri, J. D. Boeke, A. Gabriel and D. F. Voytas (1998). "Transposable elements and

genome organization: a comprehensive survey of retrotransposons revealed by the complete

Saccharomyces cerevisiae genome sequence." Genome research 8(5): 464-478.

Kleine-Vehn, J., J. Leitner, M. Zwiewka, M. Sauer, L. Abas, C. Luschnig and J. Friml (2008).

"Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting."

Proceedings of the National Academy of Sciences 105(46): 17812-17817.

References

94

Kluge, C., J. Lahr, M. Hanitzsch, S. Bolte, D. Golldack and K.-J. Dietz (2003). "New insight into the

structure and regulation of the plant vacuolar H+-ATPase." Journal of bioenergetics and

biomembranes 35(4): 377-388.

Koide, Y., K. Matsuoka, M.-a. Ohto and K. Nakamura (1999). "The N-terminal propeptide and the C

terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of

vacuolar sorting signals." Plant and Cell Physiology 40(11): 1152-1159.

Kornfeld, R. and S. Kornfeld (1985). "Assembly of asparagine-linked oligosaccharides." Annual review

of biochemistry 54(1): 631-664.

Koumandou, V. L. and C. J. Howe (2007). "The copy number of chloroplast gene minicircles changes

dramatically with growth phase in the dinoflagellate Amphidinium operculatum." Protist 158(1):

89-103.

Koumandou, V. L., R. E. R. Nisbet, A. C. Barbrook and C. J. Howe (2004). "Dinoflagellate chloroplasts–

where have all the genes gone?" TRENDS in Genetics 20(5): 261-267.

Krebs, M., D. Beyhl, E. Gorlich, K. A. Al-Rasheid, I. Marten, Y. D. Stierhof, R. Hedrich and K.

Schumacher (2010). "Arabidopsis V-ATPase activity at the tonoplast is required for efficient

nutrient storage but not for sodium accumulation." Proc Natl Acad Sci U S A 107(7): 3251-3256.

Kunnimalaiyaan, M., F. Shi and B. L. Nielsen (1997). "Analysis of the tobacco chloroplast DNA

replication origin (ori B) downstream of the 23 S rRNA gene." Journal of molecular biology 268(2):

273-283.

Laatsch, T., S. Zauner, B. Stoebe-Maier, K. V. Kowallik and U. G. Maier (2004). "Plastid-derived single

gene minicircles of the dinoflagellate Ceratium horridum are localized in the nucleus." Mol Biol

Evol 21(7): 1318-1322.

Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of

bacteriophage T4." nature 227(5259): 680-685.

Lane, C. E. and J. M. Archibald (2008). "The eukaryotic tree of life: endosymbiosis takes its TOL."

Trends Ecol Evol 23(5): 268-275.

Lee, M. C. and E. A. Miller (2007). Molecular mechanisms of COPII vesicle formation. Seminars in cell

& developmental biology, Elsevier.

Lee, M. C. and E. A. Miller (2007). "Molecular mechanisms of COPII vesicle formation." Semin Cell

Dev Biol 18(4): 424-434.

Lemgruber, L., M. Kudryashev, C. Dekiwadia, D. T. Riglar, J. Baum, H. Stahlberg, S. A. Ralph and F.

Frischknecht (2013). "Cryo-electron tomography reveals four-membrane architecture of the

Plasmodium apicoplast." Malaria journal 12(1): 25.

Li, G., V. Santoni and C. Maurel (2014). "Plant aquaporins: roles in plant physiology." Biochim

Biophys Acta 1840(5): 1574-1582.

References

95

Lienard, D., G. Durambur, M. C. Kiefer-Meyer, F. Nogue, L. Menu-Bouaouiche, F. Charlot, V. Gomord

and J. P. Lassalles (2008). "Water transport by aquaporins in the extant plant Physcomitrella

patens." Plant Physiol 146(3): 1207-1218.

Liu, L.-H., U. Ludewig, B. Gassert, W. B. Frommer and N. von Wirén (2003). "Urea transport by

nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis." Plant physiology 133(3): 1220-

1228.

Loque, D., U. Ludewig, L. Yuan and N. von Wiren (2005). "Tonoplast intrinsic proteins AtTIP2;1 and

AtTIP2;3 facilitate NH3 transport into the vacuole." Plant Physiol 137(2): 671-680.

Ma, B., D. Qian, Q. Nan, C. Tan, L. An and Y. Xiang (2012). "Arabidopsis vacuolar H+-ATPase (V-

ATPase) B subunits are involved in actin cytoskeleton remodeling via binding to, bundling, and

stabilizing F-actin." J Biol Chem 287(23): 19008-19017.

Maier, U.-G., S. E. Douglas and T. Cavalier-Smith (2000). "The nucleomorph genomes of cryptophytes

and chlorarachniophytes." Protist 151(2): 103-109.

Maldonado-Mendoza, I. E. and C. L. Nessler (1996). "Cloning and expression of a plant homologue of

the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca

acuminata." Plant Mol Biol 32(6): 1149-1153.

Mallard, F., B. L. Tang, T. Galli, D. Tenza, A. Saint-Pol, X. Yue, C. Antony, W. Hong, B. Goud and L.

Johannes (2002). "Early/recycling endosomes-to-TGN transport involves two SNARE complexes

and a Rab6 isoform." J Cell Biol 156(4): 653-664.

Marin, B., E. C. Nowack and M. Melkonian (2005). "A plastid in the making: evidence for a second

primary endosymbiosis." Protist 156(4): 425-432.

Martin, W. and R. G. Herrmann (1998). "Gene transfer from organelles to the nucleus: How much,

what happens, and why?" Plant Physiology 118(1): 9-17.

Martin, W., T. Rujan, E. Richly, A. Hansen, S. Cornelsen, T. Lins, D. Leister, B. Stoebe, M. Hasegawa

and D. Penny (2002). "Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast

genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus." Proc

Natl Acad Sci U S A 99(19): 12246-12251.

Marty, F. (1999). "Plant vacuoles." The Plant Cell 11(4): 587-599.

Maruyama, N., L. C. Mun, M. Tatsuhara, M. Sawada, M. Ishimoto and S. Utsumi (2006). "Multiple

vacuolar sorting determinants exist in soybean 11S globulin." Plant Cell 18(5): 1253-1273.

Mathieu-Rivet, E., M. C. Kiefer-Meyer, G. Vanier, C. Ovide, C. Burel, P. Lerouge and M. Bardor (2014).

"Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear

expressed biopharmaceuticals." Front Plant Sci 5: 359.

References

96

Matsuoka, K., T. Higuchi, M. Maeshima and K. Nakamura (1997). "A vacuolar-type H+-ATPase in a

nonvacuolar organelle is required for the sorting of soluble vacuolar protein precursors in tobacco

cells." The Plant Cell 9(4): 533-546.

Matsuoka, K. N. a. K. (1993). "protein targeting to the vacuole in plant cells." Plant Physiol. 101: 1-5.

Matsushima R, H. Y., Yamada K, Shimada T, Nishimura M and Nishimura IH (2003). "The ER body, a

novel endoplasmic reticulum-derived structure in arabidopsis." Plant Cell Physiol 44(7): 661-666.

Maurel, C. (1997). "Aquaporins and Water Permeability of Plant Membranes." Annu Rev Plant

Physiol Plant Mol Biol 48: 399-429.

Maurel, C. and C. Plassard (2011). "Aquaporins: for more than water at the plant-fungus interface?"

New Phytol 190(4): 815-817.

Mcfadden (2001). "Primary and secondary endosymbiosis and the origin of plastids." J phycol 37:

951-959.

McGough, I. J. and P. J. Cullen (2011). "Recent advances in retromer biology." Traffic 12(8): 963-971.

McKersie, B. D. and W. Bruce (2009). Transgenic Plants with Increased Yield, Google Patents.

McMahon, H. T. and E. Boucrot (2011). "Molecular mechanism and physiological functions of

clathrin-mediated endocytosis." Nat Rev Mol Cell Biol 12(8): 517-533.

Melkonian, M. (1996). "Phylogeny of photosynthetic protists and their plastids." VERHANDLUNGEN-

DEUTSCHEN ZOOLOGISCHEN GESELLSCHAFT 89: 71-96.

Michaeli, S., T. Avin-Wittenberg and G. Galili (2014). "Involvement of autophagy in the direct ER to

vacuole protein trafficking route in plants." Frontiers in plant science 5.

Miller, E. A., T. H. Beilharz, P. N. Malkus, M. C. S. Lee, S. Hamamoto, L. Orci and R. Schekman (2003).

"Multiple Cargo Binding Sites on the COPII Subunit Sec24p Ensure Capture of Diverse Membrane

Proteins into Transport Vesicles." Cell 114(4): 497-509.

Miyata, Y. and M. Sugita (2004). "Tissue- and stage-specific RNA editing of rps 14 transcripts in moss

(Physcomitrella patens) chloroplasts." J Plant Physiol 161(1): 113-115.

Moore, R. B. (2003). "Highly organized structure in the non-coding region of the psbA minicircle from

clade C Symbiodinium." International Journal of Systematic and Evolutionary Microbiology 53(6):

1725-1734.

Moriyasu, Y., M. Hattori, G.-Y. Jauh and J. C. Rogers (2003). "Alpha tonoplast intrinsic protein is

specifically associated with vacuole membrane involved in an autophagic process." Plant and cell

physiology 44(8): 795-802.

Mossessova, E., L. C. Bickford and J. Goldberg (2003). "SNARE Selectivity of the COPII Coat." Cell

114(4): 483-495.

Moustafa, A., B. Beszteri, U. G. Maier, C. Bowler, K. Valentin and D. Bhattacharya (2009). "Genomic

footprints of a cryptic plastid endosymbiosis in diatoms." science 324(5935): 1724-1726.

References

97

Movafeghi, A., N. Happel, P. Pimpl, G.-H. Tai and D. G. Robinson (1999). "Arabidopsis Sec21p and

Sec23p homologs. Probable coat proteins of plant COP-coated vesicles." Plant Physiology 119(4):

1437-1446.

Mungpakdee, S., C. Shinzato, T. Takeuchi, T. Kawashima, R. Koyanagi, K. Hisata, M. Tanaka, H. Goto,

M. Fujie, S. Lin, N. Satoh and E. Shoguchi (2014). "Massive gene transfer and extensive RNA

editing of a symbiotic dinoflagellate plastid genome." Genome Biol Evol 6(6): 1408-1422.

Nelson, M. J. and B. R. Green (2005). "Double hairpin elements and tandem repeats in the non-

coding region of Adenoides eludens chloroplast gene minicircles." Gene 358: 102-110.

Neuhaus, H. E. (2007). "Transport of primary metabolites across the plant vacuolar membrane."

FEBS Lett 581(12): 2223-2226.

Neuhaus, J.-M. and J. C. Rogers (1998). Sorting of proteins to vacuoles in plant cells. Protein

Trafficking in Plant Cells, Springer: 127-144.

Niemes, S., M. Langhans, C. Viotti, D. Scheuring, M. San Wan Yan, L. Jiang, S. Hillmer, D. G. Robinson

and P. Pimpl (2010). "Retromer recycles vacuolar sorting receptors from the trans-Golgi network."

Plant J 61(1): 107-121.

Nisbet, R. E., R. G. Hiller, E. R. Barry, P. Skene, A. C. Barbrook and C. J. Howe (2008). "Transcript

analysis of dinoflagellate plastid gene minicircles." Protist 159(1): 31-39.

Nisbet, R. E., L. Koumandou, A. C. Barbrook and C. J. Howe (2004). "Novel plastid gene minicircles in

the dinoflagellate Amphidinium operculatum." Gene 331: 141-147.

Nishizawa, K., N. Maruyama, R. Satoh, Y. Fuchikami, T. Higasa and S. Utsumi (2003). "AC‐terminal

sequence of soybean β‐conglycinin α′ subunit acts as a vacuolar sorting determinant in seed cells."

The Plant Journal 34(5): 647-659.

Nothwehr, S. F., P. Bruinsma and L. A. Strawn (1999). "Distinct domains within Vps35p mediate the

retrieval of two different cargo proteins from the yeast prevacuolar/endosomal compartment."

Molecular biology of the cell 10(4): 875-890.

Nowack, E. C. and A. R. Grossman (2012). "Trafficking of protein into the recently established

photosynthetic organelles of Paulinella chromatophora." Proceedings of the National Academy of

Sciences 109(14): 5340-5345.

Ochoa de Alda, J. A., R. Esteban, M. L. Diago and J. Houmard (2014). "The plastid ancestor originated

among one of the major cyanobacterial lineages." Nat Commun 5: 4937.

Ohta, N., M. Matsuzaki, O. Misumi, S.-y. Miyagishima, H. Nozaki, K. Tanaka, T. Shin-i, Y. Kohara and T.

Kuroiwa (2003). "Complete sequence and analysis of the plastid genome of the unicellular red

alga Cyanidioschyzon merolae." DNA research 10(2): 67-77.

References

98

Ohyama, K., H. Fukuzawa, T. Kohchi, H. Shirai, T. Sano, S. Sano, K. Umesono, Y. Shiki, M. Takeuchi

and Z. Chang (1986). "Chloroplast gene organization deduced from complete sequence of

liverwort Marchantia polymorpha chloroplast DNA." Nature 322: 572-574.

Oliviusson, P., O. Heinzerling, S. Hillmer, G. Hinz, Y. C. Tse, L. Jiang and D. G. Robinson (2006). "Plant

retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may

interact with vacuolar sorting receptors." Plant Cell 18(5): 1239-1252.

Otegui, M. S. and C. Spitzer (2008). "Endosomal functions in plants." Traffic 9(10): 1589-1598.

Pagny, S., F. Bouissonnie, M. Sarkar, M. Follet‐Gueye, A. Driouich, H. Schachter, L. Faye and V.

Gomord (2003). "Structural requirements for Arabidopsis β1, 2‐xylosyltransferase activity and

targeting to the Golgi." The Plant Journal 33(1): 189-203.

Pandey, B., P. Sharma, D. M. Pandey, I. Sharma and R. Chatrath (2013). "Identification of new

aquaporin genes and single nucleotide polymorphism in bread wheat." Evol Bioinform Online 9:

437-452.

Paris, N., B. Saint-Jean, M. Faraco, W. Krzeszowiec, G. Dalessandro, J.-M. Neuhaus and G. P. Di

Sansebastiano (2010). "Expression of a glycosylated GFP as a bivalent reporter in exocytosis."

Plant cell reports 29(1): 79-86.

Park, M., S. J. Kim, A. Vitale and I. Hwang (2004). "Identification of the protein storage vacuole and

protein targeting to the vacuole in leaf cells of three plant species." Plant physiology 134(2): 625-

639.

Park, W., B. E. Scheffler, P. J. Bauer and B. T. Campbell (2010). "Identification of the family of

aquaporin genes and their expression in upland cotton (Gossypium hirsutum L.)." BMC plant

biology 10(1): 142.

Patron, N. J., M. B. Rogers and P. J. Keeling (2004). "Gene replacement of fructose-1,6-bisphosphate

aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates."

Eukaryot Cell 3(5): 1169-1175.

Patron, N. J., R. F. Waller, J. M. Archibald and P. J. Keeling (2005). "Complex protein targeting to

dinoflagellate plastids." Journal of molecular biology 348(4): 1015-1024.

Pereira, C., S. Pereira and J. Pissarra (2014). "Delivering of proteins to the plant vacuole--an update."

Int J Mol Sci 15(5): 7611-7623.

Pereira, C., S. Pereira, B. Satiat-Jeunemaitre and J. Pissarra (2013). "Cardosin A contains two vacuolar

sorting signals using different vacuolar routes in tobacco epidermal cells." Plant J 76(1): 87-100.

Petersen, J., A.-K. Ludewig, V. Michael, B. Bunk, M. Jarek, D. Baurain and H. Brinkmann (2014).

"Chromera velia, endosymbioses and the rhodoplex hypothesis—plastid evolution in cryptophytes,

alveolates, stramenopiles, and haptophytes (CASH lineages)." Genome biology and evolution 6(3):

666-684.

References

99

Phan, N. Q., S.-J. Kim and D. C. Bassham (2008). "Overexpression of Arabidopsis sorting nexin

AtSNX2b inhibits endocytic trafficking to the vacuole." Molecular plant 1(6): 961-976.

Pimpl, P., S. L. Hanton, J. P. Taylor, L. L. Pinto-daSilva and J. Denecke (2003). "The GTPase ARF1p

controls the sequence-specific vacuolar sorting route to the lytic vacuole." The Plant Cell 15(5):

1242-1256.

Pimpl, P., A. Movafeghi, S. Coughlan, J. Denecke, S. Hillmer and D. G. Robinson (2000). "In situ

localization and in vitro induction of plant COPI-coated vesicles." The Plant Cell 12(11): 2219-2235.

Pimpl, P., J. P. Taylor, C. Snowden, S. Hillmer, D. G. Robinson and J. Denecke (2006). "Golgi-mediated

vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality

control within the secretory pathway." Plant Cell 18(1): 198-211.

Piper, R. C. and D. J. Katzmann (2007). "Biogenesis and function of multivesicular bodies." Annu Rev

Cell Dev Biol 23: 519-547.

Pompa, A., F. De Marchis, A. Vitale, S. Arcioni and M. Bellucci (2010). "An engineered C-terminal

disulfide bond can partially replace the phaseolin vacuolar sorting signal." Plant J 61(5): 782-791.

Popov, N., M. Schmitt, S. Schulzeck and H. Matthies (1974). "Reliable micromethod for

determination of the protein content in tissue homogenates." Acta biologica et medica Germanica

34(9): 1441-1446.

Pourcher, M., M. Santambrogio, N. Thazar, A.-M. Thierry, I. Fobis-Loisy, C. Miège, Y. Jaillais and T.

Gaude (2010). "Analyses of sorting nexins reveal distinct retromer-subcomplex functions in

development and protein sorting in Arabidopsis thaliana." The Plant Cell 22(12): 3980-3991.

Puerta, M. V. S., T. R. Bachvaroff and C. F. Delwiche (2005). "The complete plastid genome sequence

of the haptophyte Emiliania huxleyi: a comparison to other plastid genomes." DNA Research 12(2):

151-156.

Quigley, F., J. M. Rosenberg, Y. Shachar-Hill and H. J. Bohnert (2002). "From genome to function: the

Arabidopsis aquaporins." Genome Biol 3(1): 0001-0001.0017.

Rachel, R., C. Meyer, A. Klingl, S. Guerster, T. Heimerl, N. Wasserburger, T. Burghardt, U. Küper, A.

Bellack and S. Schopf (2010). "Analysis of the ultrastructure of archaea by electron microscopy."

Methods in cell biology 96: 47-69.

Regon P., Panda P., Kshetrimayum E., Panda S.K. (2014). "Genome-wide comparative analysis of

tonoplast intrinsic protein (TIP) genes in plants" 14: 617-629.

Rayon, C., P. Lerouge and L. Faye (1998). "The protein N-glycosylation in plants." Journal of

Experimental Botany 49(326): 1463-1472.

Reyes-Prieto, A., J. D. Hackett, M. B. Soares, M. F. Bonaldo and D. Bhattacharya (2006).

"Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions."

Curr Biol 16(23): 2320-2325.

References

100

Reyes-Prieto, A., A. Moustafa and D. Bhattacharya (2008). "Multiple genes of apparent algal origin

suggest ciliates may once have been photosynthetic." Curr Biol 18(13): 956-962.

Reyes, F. C., R. Buono and M. S. Otegui (2011). "Plant endosomal trafficking pathways." Curr Opin

Plant Biol 14(6): 666-673.

Reynolds, G. D., B. August and S. Y. Bednarek (2014). "Preparation of enriched plant clathrin-coated

vesicles by differential and density gradient centrifugation." Methods Mol Biol 1209: 163-177.

Rigal, A., S. M. Doyle and S. Robert (2015). Live Cell Imaging of FM4-64, a Tool for Tracing the

Endocytic Pathways in Arabidopsis Root Cells. Plant Cell Expansion, Springer: 93-103.

Ritzenthaler, C., A. Nebenführ, A. Movafeghi, C. Stussi-Garaud, L. Behnia, P. Pimpl, L. A. Staehelin

and D. G. Robinson (2002). "Reevaluation of the effects of brefeldin A on plant cells using tobacco

Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera." The

Plant Cell 14(1): 237-261.

Rivera-Serrano, E. E., M. F. Rodriguez-Welsh, G. R. Hicks and M. Rojas-Pierce (2012). "A small

molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in

Arabidopsis." PLoS One 7(9): e44735.

Robinson, D. G., G. Hinz and S. E. Holstein (1998). "The molecular characterization of transport

vesicles." Plant molecular biology 38(1-2): 49-76.

Robinson, D. G., L. Jiang and K. Schumacher (2008). "The endosomal system of plants: charting new

and familiar territories." Plant Physiology 147(4): 1482-1492.

Robinson, D. G., P. Pimpl, D. Scheuring, Y.-D. Stierhof, S. Sturm and C. Viotti (2012). "Trying to make

sense of retromer." Trends in plant science 17(7): 431-439.

Robinson, M. S. and J. S. Bonifacino (2001). "Adaptor-related proteins." Current Opinion in Cell

Biology 13(4): 444-453.

Rockwell, N. C., J. C. Lagarias and D. Bhattacharya (2014). "Primary endosymbiosis and the evolution

of light and oxygen sensing in photosynthetic eukaryotes." Front Ecol Evol 2(66).

Rodriguez-Ezpeleta, N., H. Brinkmann, S. C. Burey, B. Roure, G. Burger, W. Loffelhardt, H. J. Bohnert,

H. Philippe and B. F. Lang (2005). "Monophyly of primary photosynthetic eukaryotes: green plants,

red algae, and glaucophytes." Curr Biol 15(14): 1325-1330.

Rouillé, Y., W. Rohn and B. Hoflack (2000). Targeting of lysosomal proteins. Seminars in cell &

developmental biology, Elsevier.

Saito, C., T. Ueda, H. Abe, Y. Wada, T. Kuroiwa, A. Hisada, M. Furuya and A. Nakano (2002). "A

complex and mobile structure forms a distinct subregion within the continuous vacuolar

membrane in young cotyledons of Arabidopsis." The Plant Journal 29(3): 245-255.

Sanger, F., S. Nicklen and A. R. Coulson (1977). "DNA sequencing with chain-terminating inhibitors."

Proceedings of the National Academy of Sciences 74(12): 5463-5467.

References

101

Sanmartin, M., A. Ordonez, E. J. Sohn, S. Robert, J. J. Sanchez-Serrano, M. A. Surpin, N. V. Raikhel and

E. Rojo (2007). "Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles

in Arabidopsis." Proc Natl Acad Sci U S A 104(9): 3645-3650.

Santos, S. R., D. J. Taylor, R. A. Kinzie Iii, M. Hidaka, K. Sakai and M. A. Coffroth (2002). "Molecular

phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA

sequences." Molecular phylogenetics and evolution 23(2): 97-111.

Schüssler, M. D., E. Alexandersson, G. P. Bienert, T. Kichey, K. H. Laursen, U. Johanson, P. Kjellbom, J.

K. Schjoerring and T. P. Jahn (2008). "The effects of the loss of TIP1; 1 and TIP1; 2 aquaporins in

Arabidopsis thaliana." The Plant Journal 56(5): 756-767.

Schellmann, S. and P. Pimpl (2009). "Coats of endosomal protein sorting: retromer and ESCRT." Curr

Opin Plant Biol 12(6): 670-676.

Scheuring, D., C. Viotti, F. Krüger, F. Künzl, S. Sturm, J. Bubeck, S. Hillmer, L. Frigerio, D. G. Robinson

and P. Pimpl (2011). "Multivesicular bodies mature from the trans-Golgi network/early endosome

in Arabidopsis." The Plant Cell 23(9): 3463-3481.

Schleiff, E. and T. Becker (2011). "Common ground for protein translocation: access control for

mitochondria and chloroplasts." Nature Reviews Molecular Cell Biology 12(1): 48-59.

Schnable, P. S., D. Ware, R. S. Fulton, J. C. Stein, F. Wei, S. Pasternak, C. Liang, J. Zhang, L. Fulton and

T. A. Graves (2009). "The B73 maize genome: complexity, diversity, and dynamics." science

326(5956): 1112-1115.

Schoberer, J., U. Vavra, J. Stadlmann, C. Hawes, L. Mach, H. Steinkellner and R. Strasser (2009).

"Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation

enzymes." Traffic 10(1): 101-115.

Seaman, M. N. (2004). "Cargo-selective endosomal sorting for retrieval to the Golgi requires

retromer." The Journal of cell biology 165(1): 111-122.

Seaman, M. N. (2005). "Recycle your receptors with retromer." Trends Cell Biol 15(2): 68-75.

Seaman, M. N., M. E. Harbour, D. Tattersall, E. Read and N. Bright (2009). "Membrane recruitment of

the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by

the Rab-GAP TBC1D5." Journal of cell science 122(14): 2371-2382.

Sears, B. B., L. L. Stoike and W.-L. Chiu (1996). "Proliferation of direct repeats near the Oenothera

chloroplast DNA origin of replication." Molecular biology and evolution 13(6): 850-863.

Seidel, T., D. Schnitzer, D. Golldack, M. Sauer and K. J. Dietz (2008). "Organelle-specific isoenzymes of

plant V-ATPase as revealed by in vivo-FRET analysis." BMC Cell Biol 9: 28.

Shalchian-Tabrizi, K., M. Skånseng, F. Ronquist, D. Klaveness, T. R. Bachvaroff, C. F. Delwiche, A.

Botnen, T. Tengs and K. S. Jakobsen (2006). "Heterotachy processes in rhodophyte-derived

References

102

secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate

plastids." Molecular biology and evolution 23(8): 1504-1515.

Shimada, T., K. Fuji, K. Tamura, M. Kondo, M. Nishimura and I. Hara-Nishimura (2003). "Vacuolar

sorting receptor for seed storage proteins in Arabidopsis thaliana." Proc Natl Acad Sci U S A

100(26): 16095-16100.

Shinozaki, K., M. Ohme, M. Tanaka, T. Wakasugi, N. Hayashida, T. Matsubayashi, N. Zaita, J.

Chunwongse, J. Obokata and K. Yamaguchi-Shinozaki (1986). "The complete nucleotide sequence

of the tobacco chloroplast genome: its gene organization and expression." The EMBO journal 5(9):

2043.

Simoes, I. and C. Faro (2004). "Structure and function of plant aspartic proteinases." Eur J Biochem

271(11): 2067-2075.

Sommer, M. S., S. B. Gould, P. Lehmann, A. Gruber, J. M. Przyborski and U. G. Maier (2007). "Der1-

mediated preprotein import into the periplastid compartment of chromalveolates?" Mol Biol Evol

24(4): 918-928.

Song, J., M. H. Lee, G. J. Lee, C. M. Yoo and I. Hwang (2006). "Arabidopsis EPSIN1 plays an important

role in vacuolar trafficking of soluble cargo proteins in plant cells via interactions with clathrin, AP-

1, VTI11, and VSR1." Plant Cell 18(9): 2258-2274.

Stegemann, S., S. Hartmann, S. Ruf and R. Bock (2003). "High-frequency gene transfer from the

chloroplast genome to the nucleus." Proc Natl Acad Sci U S A 100(15): 8828-8833.

Stierhof, Y.-D., C. Viotti, D. Scheuring, S. Sturm and D. G. Robinson (2013). "Sorting nexins 1 and 2a

locate mainly to the TGN." Protoplasma 250(1): 235-240.

Stoebe, B. and U. G. Maier (2002). "One, two, three: nature's tool box for building plastids."

Protoplasma 219(3-4): 123-130.

Stork, S. (2013). "Investigation of the proteome of the periplastidal membrane and of Sec61-

dependent membrane protein import into the complex plastid of the diatom Phaeodactylum

tricornutum." Dissertation.

Stork, S., J. Lau, D. Moog and U. G. Maier (2013). "Three old and one new: protein import into red

algal-derived plastids surrounded by four membranes." Protoplasma 250(5): 1013-1023.

Strasser, R. (2014). "Biological significance of complex N-glycans in plants and their impact on plant

physiology." Front Plant Sci 5: 363.

Strasser, R., J. S. Bondili, U. Vavra, J. Schoberer, B. Svoboda, J. Glössl, R. Léonard, J. Stadlmann, F.

Altmann and H. Steinkellner (2007). "A unique β1, 3-galactosyltransferase is indispensable for the

biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana." The Plant Cell

19(7): 2278-2292.

References

103

Strasser, R., J. Mucha, L. Mach, F. Altmann, I. Wilson, J. Glössl and H. Steinkellner (2000). "Molecular

cloning and functional expression of β1, 2-xylosyltransferase cDNA from Arabidopsis thaliana."

Febs Letters 472(1): 105-108.

Swanson, S. J., P. C. Bethke and R. L. Jones (1998). "Barley aleurone cells contain two types of

vacuoles: characterization of lytic organelles by use of fluorescent probes." The Plant Cell 10(5):

685-698.

Sze, H., K. Schumacher, M. L. Müller, S. Padmanaban and L. Taiz (2002). "A simple nomenclature for

a complex proton pump: VHA genes encode the vacuolar H+-ATPase." Trends in plant science 7(4):

157-161.

Takenaka, M., A. Zehrmann, D. Verbitskiy, B. Hartel and A. Brennicke (2013). "RNA editing in plants

and its evolution." Annu Rev Genet 47: 335-352.

Takeuchi, M., T. Ueda, N. Yahara and A. Nakano (2002). "Arf1 GTPase plays roles in the protein traffic

between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured

cells." The Plant Journal 31(4): 499-515.

Takishita, K., K.-i. Ishida and T. Maruyama (2004). "Phylogeny of nuclear-encoded plastid-targeted

GAPDH gene supports separate origins for the peridinin-and the fucoxanthin derivative-containing

plastids of dinoflagellates." Protist 155(4): 447-458.

Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar (2013). "MEGA6: molecular evolutionary

genetics analysis version 6.0." Molecular biology and evolution 30(12): 2725-2729.

Tanikawa, N., H. Akimoto, K. Ogoh, W. Chun and Y. Ohmiya (2004). "Expressed Sequence Tag

Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase." Photochemistry and

photobiology 80(1): 31-35.

Teich, R., S. Zauner, D. Baurain, H. Brinkmann and J. Petersen (2007). "Origin and distribution of

Calvin cycle fructose and sedoheptulose bisphosphatases in plantae and complex algae: a single

secondary origin of complex red plastids and subsequent propagation via tertiary

endosymbioses." Protist 158(3): 263-276.

Tse, Y. C., B. Mo, S. Hillmer, M. Zhao, S. W. Lo, D. G. Robinson and L. Jiang (2004). "Identification of

multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells." Plant Cell

16(3): 672-693.

Uehlein, N. and R. Kaldenhoff (2008). "Aquaporins and plant leaf movements." Ann Bot 101(1): 1-4.

Uenishi, Y., Y. Nakabayashi, A. Tsuchihira, M. Takusagawa, K. Hashimoto, M. Maeshima and K. Sato-

Nara (2014). "Accumulation of TIP2; 2 aquaporin during dark adaptation is partially PhyA

dependent in roots of Arabidopsis seedlings." Plants 3(1): 177-195.

References

104

Van Damme, D., A. Gadeyne, M. Vanstraelen, D. Inze, M. C. Van Montagu, G. De Jaeger, E. Russinova

and D. Geelen (2011). "Adaptin-like protein TPLATE and clathrin recruitment during plant somatic

cytokinesis occurs via two distinct pathways." Proc Natl Acad Sci U S A 108(2): 615-620.

Vergés, M., I. Sebastián and K. E. Mostov (2007). "Phosphoinositide 3-kinase regulates the role of

retromer in transcytosis of the polymeric immunoglobulin receptor." Experimental cell research

313(4): 707-718.

Viotti, C., J. Bubeck, Y. D. Stierhof, M. Krebs, M. Langhans, W. van den Berg, W. van Dongen, S.

Richter, N. Geldner, J. Takano, G. Jurgens, S. C. de Vries, D. G. Robinson and K. Schumacher (2010).

"Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome,

an independent and highly dynamic organelle." Plant Cell 22(4): 1344-1357.

Vitale, A. and G. Hinz (2005). "Sorting of proteins to storage vacuoles: how many mechanisms?"

Trends Plant Sci 10(7): 316-323.

Vitale, H. H. a. A. (2001). "Vacuolar sorting determinants within a plant storage protein trimer act

cumulatively " traffic 2: 737-741.

Wang, Y., L. Jensen, P. Højrup and D. Morse (2005). "Synthesis and degradation of dinoflagellate

plastid-encoded psbA proteins are light-regulated, not circadian-regulated." Proceedings of the

National Academy of Sciences of the United States of America 102(8): 2844-2849.

Wang, Y. and D. Morse (2006). "Rampant polyuridylylation of plastid gene transcripts in the

dinoflagellate Lingulodinium." Nucleic Acids Res 34(2): 613-619.

Watanabe, E., T. Shimada, K. Tamura, R. Matsushima, Y. Koumoto, M. Nishimura and I. Hara-

Nishimura (2004). "An ER-localized form of PV72, a seed-specific vacuolar sorting receptor,

interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves." Plant and cell

physiology 45(1): 9-17.

Weber, A. P., M. Linka and D. Bhattacharya (2006). "Single, ancient origin of a plastid metabolite

translocator family in Plantae from an endomembrane-derived ancestor." Eukaryot Cell 5(3): 609-

612.

Werner, M., N. Uehlein, P. Proksch and R. Kaldenhoff (2001). "Characterization of two tomato

aquaporins and expression during the incompatible interaction of tomato with the plant parasite

Cuscuta reflexa." Planta 213(4): 550-555.

Wilson, I. B. (2002). "Glycosylation of proteins in plants and invertebrates." Current opinion in

structural biology 12(5): 569-577.

Wudick, M. M., D. T. Luu, C. Tournaire-Roux, W. Sakamoto and C. Maurel (2014). "Vegetative and

sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and

contribute to plant reproduction." Plant Physiol 164(4): 1697-1706.

Xi, C. and J. Wu (2011). Control of biofilm formation, Google Patents.

References

105

Xiang, L., E. Etxeberria and W. Van den Ende (2013). "Vacuolar protein sorting mechanisms in

plants." FEBS J 280(4): 979-993.

Xiang, L. and W. Van den Ende (2013). "Trafficking of plant vacuolar invertases: from a membrane-

anchored to a soluble status. Understanding sorting information in their complex N-terminal

motifs." Plant Cell Physiol 54(8): 1263-1277.

Yamazaki, M., T. Shimada, H. Takahashi, K. Tamura, M. Kondo, M. Nishimura and I. Hara-Nishimura

(2008). "Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and

involved in plant growth and leaf senescence." Plant and cell physiology 49(2): 142-156.

Yang, Y. D., R. Elamawi, J. Bubeck, R. Pepperkok, C. Ritzenthaler and D. G. Robinson (2005).

"Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells

provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites."

Plant Cell 17(5): 1513-1531.

Zardoya, R. (2005). "Phylogeny and evolution of the major intrinsic protein family." Biology of the

Cell 97(6): 397-414.

Zaslavskaia, L. A., J. C. Lippmeier, P. G. Kroth, A. R. Grossman and K. E. Apt (2000). "Transformation

of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker

and reporter genes." Journal of Phycology 36(2): 379-386.

Zauner, S., D. Greilinger, T. Laatsch, K. V. Kowallik and U. G. Maier (2004). "Substitutional editing of

transcripts from genes of cyanobacterial origin in the dinoflagellate Ceratium horridum." FEBS Lett

577(3): 535-538.

Zhang, C., G. R. Hicks and N. V. Raikhel (2014). "Plant vacuole morphology and vacuolar trafficking."

Front Plant Sci 5: 476.

Zhang, Z., T. Cavalier-Smith and B. R. Green (2002). "Evolution of dinoflagellate unigenic minicircles

and the partially concerted divergence of their putative replicon origins." Mol Biol Evol 19(4): 489-

500.

Zhang, Z., B. Green and T. Cavalier-Smith (1999). "Single gene circles in dinoflagellate chloroplast

genomes." Nature 400(6740): 155-159.

Zhang, Z., B. R. Green and T. Cavalier-Smith (2000). "Phylogeny of ultra-rapidly evolving

dinoflagellate chloroplast genes: a possible common origin for sporozoan and dinoflagellate

plastids." Journal of Molecular Evolution 51(1): 26-40.

Zhou, A., Y. Bu, T. Takano, X. Zhang and S. Liu (2015). "Conserved V‐ATPase c subunit plays a role in

plant growth by influencing V‐ATPase‐dependent endosomal trafficking." Plant biotechnology

journal.

Zimorski, V., C. Ku, W. F. Martin and S. B. Gould (2014). "Endosymbiotic theory for organelle origins."

Current opinion in microbiology 22: 38-48.

References

106

Zouhar, J. and E. Rojo (2009). "Plant vacuoles: where did they come from and where are they

heading?" Curr Opin Plant Biol 12(6): 677-684.

Supplements

107

7 Supplements

7.1 Open reading frames with no known homology

Table S1: All open reading frames mentioned in this study.

Only the open reading frames >= 150 bps were listed in this table. J8-, J9-, J13-, J22-, J24-, J36- ORFs are the open reading

frames of empty minicircles in A. carterae CCAM0512. J28 ORFs are the open reading frames of atpB gene containing

minicircle in A. carterae CCAM0512. The rests are the open reading frames of empty minicircles in A. carterae CCAP 1102/6

and A. carterae CS21, they are named by the accession numbers plus the ORF number in the table.

name Open reading frames

J8 ORF1 MGDPNIDSMAFNHREGLRYQKCCLRVTKNSGLHHLIGSKEDDHIALQLRSQLSN.

J8 ORF2 MHELVVANTTSPLTTFDRGGFALKQAYSIRGPDPAGVEEHTTRIELLKRTIVLISNYSMIANYA.

J8 ORF3 MAVRQIQLPSGTALIGKGRGCNTAQVITFVRRHTTTNIATLNRWRCPCRAQEGRKTWPFYQ.

J8 ORF4 MTRHHYTELVKSKRQHRCTPPNKQGNYSFSIPTKLEGVRGSTFRPLHSYLPQRRGRDRSLILAYPQNEARTINLEPNHRVLN.

J9 ORF1 MPRHLPSVGASFLQARLDRSLDSRCTMVSLNDVTSNGLRVLAYFPLNDSTMAGNNVSWSIST.

J9 ORF2 MLSLFVVIIRCHAIKEDRTMLALMPFGLRVYTVVRRLPCPVSSDKGGYERLSCSNNSNLVPALRTNF.

J9 ORF3 MSNLLAISVVSVTISRPLGPNWILSLIHISTIIDEFSRVFSHIGSNSSSVPGDPLESTCRGGGESHVVSQNL.

J9 ORF4 MIYFYLVQCNIRDFETQRGFPPPPCRSTLEDPRVPSSNSSQYARTPEKIHR.

J9 ORF5 MVEMCIRDRIQLGPRGRDIVTDTTLIARRLLIHYRQQLSLHRRGNYVGLQTSDCRRHRPHLSMC.

J9 ORF6 MYLPSSTVLYLGAFHLICLSISVVSTFIFVPSSTFLFDPSELSGESPDLCANYHLIPFTSSSSSFSRSHHSHSYGLTGIF.

J9 ORF7 MIVSRSTKNLCVKRVRGWSCCCRIVFHNLPCQKTQGKVDVLQQCRHEDQMALEPTSFYPL.

J9 ORF8 MQTGIEDVYHFLMKVNTCYSVIEITNGLHSIGQEYSWLVLRYQDRYPGVQHTQ.

J9 ORF9 MATSLITCHQTREDIIDPIRTKRTRYRHRHDTDSEEVAHSLSPTTLIASSR.

J13 ORF1 MEYVEWLSSNATGMACWLVWRCRSRVMMPCLQGCYPSHVVAYQSRTINSCE.

J13 ORF2 MLLRSYLAKHSLRRYVNSLGLPISYTEPGTVLVSSSSFLLRKPKYGLVRLASLNPTLALNVVQVNALNHAALLSNSLVRSIEDGCNPTIVFQSVMGTLKRSNRVLGVKLELRALVLAPFARHRVWNYGYGKTNKAVARIKDVGKASLVTYMGLISVSTAISSG.

J13 ORF3 MSPQRSIAPFQVICLSAPLPSIEGLSLSFHQLSLHSFVYSLVEILTSRHI.

J22 ORF1 MSHDIITTTPNPLSFIGGGLIKVKSLWPMRGAIHQLQYLVHSPSYRRSSEH.


J22 ORF3 MVLKNIPDCTNKVTDRHSIVPAYQDEYLDPSTKHEPRVPFLTSGVNLTRYKDATSVLWQLARCD.

J24 ORF1 MLIVLTCVPFREVYATYLFQFLYGSHYSLLHGNSVPLVYVISITSVALTEDCLSINI.

J24 ORF2 MNDITNFIRVLNSYSTFSLEAFVPCVELPDPRLNSTNRSFSRHSVFVLVCDFLTPMD.

J24 ORF3 MDNMMSCLVGASAYPHWQLSQSGSPLCVKTTRFLLIPRTVYLNWWYYGTVGL.

J24 ORF4 MSHDIITTTPNPLSFIGGGLIKVKSLWPMRGAIHQLQYLVHSPSYRRSSEH.

J24 ORF5 MNDILGNLKLTDNRFHKDTSFKKCRSIHRSEKITDENEDRMTGERTISRVKTWIWQFYTRHKGLQRERRIRIQDSNKVSYVVHTDRV.

J24 ORF6 MSSDGETELHHVWEDVYDTLHPVVTIQDVPAVAVIHLWQALCNVIRCKAGLGINP.


J36 ORF2 MGRDTHLGKRVQWNAYPSPCWCNQVCIFRTVQLYESSYSVNCDQRYWSSVSSFPSPYGTVGVVRR.

J28 ORF1 MIYFYLVQCNIRDFETQRGFPPPPCRSTLEDPRVPSSNSSQYARTPEKIHR.

J28 ORF2 MRDGSVLASECEAFVWDLFYMESFVSMLHSVFKGIAPTPNKEALDLISLELEYCASNELSLALASSGLFIKSYANALIAEVQQIAYGGILRAVALAGTDGLDLVSTYGHLTYQPLVVPVGRVCQGRILNCVGAPMDAYDDIVISAAYSSVESPASVVLNALWYGGTASSSPSKLTTPLAHYDQSFANAAPIHKLSLIHISTIIDEFSRVFSHIGSNSSSVPGDPLESTCRGGGESHVVSQNL.

AJ307015 ORF1

MHELVVANTTSPLTTFDRGGFALKQAYSIRGLLSQRDQRGAYHSH.

AJ582641 ORF1

MAVRQIQLPSGTALIGKGL.

Supplements

108

DQ507216 ORF1

MLLRSYLAKHSLRRYVNSLGLPISFLPGTVLVSSSSFLLRKPKYGLVRLPLSTRLFHPMSVQVNALNHAALLSNSLVRSIEGGCNPTIVFQSVMGTLKRSNRVLGVKLELRALVLAPFARHRVWNYGYGKTNKAVARIKDVGKASLVTYMGLISVSTAISSG.

AF401630 ORF1

MSHDIITTTPIPLSFIGGGLIKVKSLWPMRGAIHQLQYLVHSPSYRRSSEH.

AF401630 ORF2

MNDITNFIRVLNSYFTFLLEAFKPCVELPDPRLNSI.

AJ318067 ORF1

MNDITNFIRVLNSYSTFLLEASEPCVELLDPRS.

AJ318067 ORF2

MSSDSETELHHVWEDVYDTLHPLVTIQDVPAVAVIHLWQALCNVIRCKAGLGINPSISTRPSWALQGSL.

7.2 Identified marker proteins in P. tricornutum

Table S2: Proteins localized as eGFP/ mRFP fusions in this study.

Protein ID from http://genome.jgi-psf.org/Phatr2/Phatr2.home.html. “-” means unknown. Abbreviations: eGFP, enhanced

green fluorescent protein; mRFP, monomeric red fluorescent protein; Tip1-5, Tonoplast intrinsic protein 1-5; GnTI, N-

acetylglucosaminyltransferase I; XylT, β1,2-xylosyltransferase; FucT, α1,3-fucosyltransferase; Vps26, Vacuolar protein

sorting 26; Vps29, Vacuolar protein sorting 29; ATPase1-2, Vacuolar type H+-ATPase 1-2.

Pt-Protein ID eGFP/ mRFP fusion protein Potential compartment

31553 Tip1-eGFP cytoplasmic membrane, endocytic vesicles

- Tip2-eGFP Vacuolar-like membrane

20755 Tip3-eGFP/ mRFP ER membrane (cER, nuclear envelope, host ER)

19409 Tip4-eGFP periplastidal membrane

43157 Tip5-eGFP ER membrane (cER, nuclear envelope, host ER)

54844 GnT1-eGFP cis Golgi

45496 XylT-eGFP/ mRFP medial Golgi

54599 FucT-eGFP Trans golgi network

41962 Vps26-eGFP Trans golgi network

17936 Vps29-eGFP/ mRFP Trans golgi network

21882 ATPase1-eGFP lytic vacuole? A second type of vacuole

44050 ATPase2-eGFP cytosol

7.3 The Tip2 eGFP fusion protein

The gene model of Tip2 is not supported completely by ESTs, so the gene was amplified from cDNA.

The gene model and all ESTs were aligned by ClustalX2.1 (Fig. S1). Together with this the protein

sequence of Tip2 is different from the genome database (Protein ID: 1370), the sequence was shown

[1].

http://genome.jgi-psf.org/Phatr2/Phatr2.home.html

Supplements

109

Fig. S1: The alignment of Tip2 nucleotide sequences in P. tricornutum.

It was shown that the 5’ terminus of Tip2 gene model is not completely supported by ESTs. Tip2 gene was amplified from

cDNA. Only the part is not completely supported by ESTs was shown in this figure. The protein sequence of Tip2_FL_eGFP

was shown [1].

[1] Tip2-eGFP fusion protein sequence used for in vivo localization studies in P. tricornutum.

>Tip2-eGFP

MVDSKSIKDPESQGYGSIQGASSHYASTEHTVEEADAEEGPFPVDLKSMLIAEVFGTCTFVQIGCAANAVALYTHN

STTMTIDWQVGVVWALAMTVAVFLSAALSGAHLNPAVSFSFALARPADFRFRKLIPYWAAQLGGALLAGIINLFLF

HQAISHYEKKMAIVPGAAGSIQSAAAFGCYWSLNSKYISNGVHAFFIEAFGTGVLVFCIFAATHIKNPLPGVAVPPII

GAMYGILVVTLGPMTGGSFNPVRDMGPRIVSVIGHWGPTALTNFLPYLLGPMIGGPIGAFLADKVLML:eGFP.

7.4 Sequences of all used oligonucleotides

Cutting sites of restriction enzymes are marked in red.

Protein name Primer name sequence

XylT Golgi pro-MunI-for CAATTGATGGCGTTTTTGCCGAATCG

Golgi pro-BglII-rev AGATCTGAATAAGGTACTCTTAATTGTCC

Vps26 Vps26-MunI-for CAATTGATGAACGTTGGATCTTTACTTGG

Vps26-BglII-rev AGATCTCCCCAAGTCTTTCCGCCATAAG

Vps29 Vps29-EcoRI-for GAATTCATGGCCAATTTTGGGGAGCTTG

Vps29-BglII-rev AGATCTTGTCAAGAGCGAAGCCATAAGG

v-ATPases1 v-ATPases1-for GAATTCATGAGTGTCGAAATGGAAACTTGC

v-ATPases1-rev GGATCCGTTGTTCCCCTCGCACACGAA

v-ATPases2 v-ATPases2-EcoRI-for GAATTCATGGCGGAATCAGGTACCGGC

v-ATPases2-BamHI-rev GGATCCGACCGTCCCGGCCATCTTTTGCG

Supplements

110

TIP1 TIP1-for1-Xba1 TCTAGAATGGCTTCCATTATCAACAT

TIP1-rev1-BamHI GGATCCGGCCTGAGGCGCGGCGTTTTCC

TIP2 EcoRI-TIP2-for GAATTCATGGTAGATTCAAAGTCAATCA

XbaI-TIP2-rev TCTAGATAGCATTAGCACTTTGTCGGCC

TIP3 EcoRI-TIP3-for2 GAATTCATGGTTGAGTACGGTGAGTTCGC

TIP3/Xba1-rev TCTAGACGCCATGAGCAGGCGGTCCGC

TIP4 Aqua2_3_BglII_neu AGATCTGGCGCCACCGTACAAAAC

Aqua2_5_EcoRI GAATTCATGGGGCGCCGTTGGTTG

TIP5 TIP5-for1-Xba1 TCTAGAATGGTCAAGGACTACGTCGAAGC

TIP5-rev1-BamHI GGATCCGTTAGTCTTCTTGGCAGTGGTC

GnT1 GnT1-for-MunI CAATTGATGCGGTTGTGGAAACG

GnT1-rev-BamHI GGATCCTCTTTTCGGTGACGGAA

FucT FucT-for-SacI GAGCTCATGTCACTTCGCAAG

FucT-rev-BglII AGATCTCGGATCGAACTTCCA

SA-GFP colony PCR

P.t.MGDG1_Bam_r GGATCCGGCACTCCCGAGATCAGTG

PDI_rv_BglII AGATCTCAATTCGCCTTCATCAAAAAGATCC

pJet seq pJet-uni CTCTCAAGATTTTCAGGCTGTAT

pJet-rev GCACAAGTGTTAAAGCAGTT

pPha-Dual seq pPha_5'vorNdeI GCTTAACTATGCGGCATCAG

pPhaDual_seq_MCS_EcoHind_for GGACATATTGTCGTTAGAACGCGG

pPhaDual_seq_MCS_EcoHind_rev GTCTTATCCAGGTCCAAACAGATTG

pPhaDual_seq_MCS_SpeSac_rv CTAACGCAGCTTAGACATAAAC

pPha-NR seq pPha-NR-for GGTCGGGTTTCGGATCCTTCC

pPha-NR-rev GATGAACATAAAACGACGATGAG

eGFP eGFP-for-BamHI GGATCCATGGTGAGCAAGGGCGAG

eGFP-rev-HindIII AAGCTTTTACCTGTACAGCTCGTCCATG

eGFP-for-XbaI TCTAGAATGGTGAGCAAGGGCGAG

eGFP-rev-5’ CGTCTCCCATGGCTCTGATTTCCCGATTTGG

Sa-GFP Sa-GFP-1-10-fw-EcoRI GAATTCATGGGTGGCACTAGTAGC

Sa-GFP-1-10-rv-BamHI GGATCCGGTACCCTTTTCGTTGGG

Sa-GFP-11-fw-SpeI ACTAGTCGTACTGGGCGAAAGC

Sa-GFP-11-rv-XbaI TCTAGAGGATCCGCCACCAGACC

Minicircles Transposon forward PCR ATTCAGGCTGCGCAACTGT

Transposon reverse PCR GTCAGTGAGCGAGGAAGCGGAAG

J25-rev2-primer GCTGCCAATGAGGGTACGCGGA

J25-for2-primer TCCGCGTACCCTCATTGGCAGC

J30i2 CAAGATCAATAAATTAGTAATGGTG

J14i2 GTTGTTTCCTTATTTGAGGGGCAG

J33i2 CGCAACCAGATATTAGCCTATACG

petD_for AC CCCTTTTGGATTAATGGTTG

petD_for2 AC TCAGGACATATTGGTCTTCC

petD_rev AC AAGTAGCATTACACGAATGG

atpB rev out AC TGTAGAATAGGTCCCAGACG

atpB for out AC TGATGGTATCCTTACAGGTC

atpB for2 AC AACCTTCGGTCTATGACTCC

Minicircles core out AC_for2 ACTCCGGGTCAATCGTTTCC

petB_for2_AC TAGGCCAGAAGTATACCAGG

petB_rev2_AC ATAATGCCTCGGGAGAATAG

petB_for out_AC TTTCCTAATGATTCGTAAGC

AC_T5_for ATACCCTTCGTTATCCTTCG

AC_T6_for TTGCTGCGTTCGTATCTTGC

AC_T10_for TTTAACACCTTTCGCCCTCG

AC_T3_for AAACACTCGTGCCCCATCAG

AC_T8_for AAGAGGATTAGGGGTTGTGG

AC_T16_for TTACGCGATACAGCTCATCC

Supplements

111

AC_T14_for ATCGTGGATGAGCTGTATCG

core out AC_rev TGGAAACGATTGTCGGTGAG

AC_T13_for TGTGATGAGGTCTGTAGTGG

AC_T13_rev TGTGATGAGGTCTGTAGTGG

TP sequencing-for CGTACTATCAACAGGTTGAACTGCC

TP sequencing-rev GAGCCAATATGCGAGAACACCCGAG

AC_T12_for ACTCGATGACCTCAACCTTG

AC_T12_rev TGATGAGGTGCCTGACAAGC

AC_T17_for ACCTGATTGCACCAACAAGG

AC_T17_rev ATGGGATTGTATCAGGGGAC

J12-sequencing primer for1 GTTCCCAGATAAGGGAATTAGGGTTC

J12-sequencing primer rev1 CTCATCGAAGACAGCGGTAGACG

psaB-rev-AC TTGAAGGAGAGATCCATACC

J2-sequencing primer for1 GCAGAGACTGCTGGTTCTGAGTCCCT

J2-sequencing primer rev1 CCCTTGTATTACTGTTTATGTAAGC

J28i3 primer AACCTTCGGTCTATGACTCC

J32i primer for ATAATGCCTCGGGAGAATAG

J32i primer rev TAGGCCAGAAGTATACCAGG

Acknowledgements

112

8 Acknowledgements

First and foremost, I would like to express my deepest gratitude to Dr. Stefan Zauner and Prof. Dr.

Uwe G. Maier for giving me the Ph.D position, for their excellent guidance, caring, patience, and

providing me with an excellent platform for finishing the Ph.D study. I would never have been able

to finish my research and writing without them. Besides my supervisors, I would also like to thank

the rest of my committee members Prof. Ralf Jacob and Prof. Andrea Maisner for their insightful

comments and encouragement for the past several years.

I gratefully acknowledge financial support from the Deutsche Forschungsgemeischaft in the RTG

1216 “Intra- and Intercellular Transport and Communication” (IITC).

I would like to thank Prof. Muriel Bardor and Clément Ovide for friendly giving me two constructs

(GnTI and FucT). I would also like to thank Dr. Thomas Heimerl and Dr. Kathrin Bolte for doing the

work on the electron microscope.

Thanks to Heidi Thierfelder for her constant willingness to culture dinoflagellates for me. Thanks to

Lucette Claudet for her friendly help in dealing with administrative stuff.

Furthermore, I would like to thank Dr. Christopher Grosche, Dr. Simone Stork and Dr. Julia Lau for

giving me help and suggestions in lab. I would also like to thank Dr. Stefan Zauner, Dr. Christopher

Grosche, Dr. Julia Lau, Jonny Gentil, Viktoria Schreiber and Christian Mayer who patiently corrected

my writing. Without them this work would not have been possible.

Special thanks goes to Angela Zimmer, Kamilla, Jonny Gentil, Dr. Simone Stork, Eike M. Trapp, Dr.

Julia Lau and all other students of AG Maier for the help in my German life.

Last but not the least, I would like to thank my family: my parents, my brother and my husband for

the unceasing encouragement, attention, love and support.

Curriculum vitae

113

9 Curriculum vitae

Personal Data:

Name: Xiaojuan Liu

Born: 12/07/1985

In: Wuping, Longyan, Fujian, China

Education:

09/1993-06/1999 Primary school

09/1999-06/2002 Middle school

09/2002-06/2005 High school

09/2005-06/2009 Fujian Normal University Biology Department Bachelor thesis in the lab of Prof. Yi Zheng

09/2009-06/2012

Fujian Normal University Biology Department, Aquatic Biology Master thesis in the lab of Prof. Huiru Zhuang

09/2012-10/2015

Philipps University of Marburg Biology Department, Cell Biology Ph.D. thesis in the lab of Prof. Dr. Uwe G. Maier and Dr. Stefan Zauner

Erklärung

114

10 Erklärung

Ich versichere, dass ich meine Dissertation

“Genetic compartmentalization in the complex plastid of Amphidinium carterae and The

endomembrane system (ES) in Phaeodactylum tricornutum”

Selbständig, ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir

ausdrücklich beyeichneten Quellen und Hilfen bedient habe. Die Dissertation wurde in der jetzigen

oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht und hat noch keinen

sonstigen Prüfungszwecken gedient.

Marburg, 30/08/2015 Xiaojuan Liu

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